Inhibition of expansion and function of pathogenic age-associated B cells and use for the prevention and treatment of autoimmune disease

ABSTRACT

This current invention provides methods and agents for preventing and treating autoimmune and lymphoproliferative disease by targeting pathogenic age-associated B cells as well as methods of detecting these pathogenic age-associated B cells as a method of diagnosing and predicting autoimmune disease and other lymphoproliferative and chronic inflammatory disorders.The current invention also provides targets for drug development and basic research for autoimmune diseases and other lymphoproliferative and chronic inflammatory disorders.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. application Ser. No.16/523,055, filed Jul. 26, 2019, which is a continuation of U.S.application Ser. No. 15/833,398, filed Dec. 6, 2017, and claims priorityto U.S. Patent Application Ser. No. 62/430,732 filed Dec. 6, 2016, U.S.Patent Application Ser. No. 62/487,645 filed Apr. 20, 2017, and U.S.Patent Application Ser. No. 62/512,803 filed May 31, 2017, all of whichare hereby incorporated by reference in their entirety.

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under AR064883 andAR070146 awarded by NIH. The government has certain rights in theinvention.

FIELD OF THE INVENTION

This invention relates to the field of preventing and treatingautoimmune and lymphoproliferative disease by targeting pathogenicage-associated B cells as well as methods of detecting these pathogenicage-associated B cells as a method of diagnosing and predictingautoimmune disease and other lymphoproliferative and chronicinflammatory disorders.

The invention also provides targets for drug development and basicresearch for autoimmune diseases and other lymphoproliferative andchronic inflammatory disorders.

BACKGROUND OF THE INVENTION

Autoimmune diseases, such as systemic lupus erythematosus (SLE), alsoknown as lupus, occur when the immune system of a vertebrate attacks thetissue of self rather than an infectious agent. Other autoimmuneillnesses include rheumatoid arthritis, inflammatory bowel disease, andType 1 diabetes. Autoimmune disorders can furthermore be associated withthe development of lymphoproliferative disorders such as lymphomas.

While the cause of lupus and other autoimmune diseases is unknown,theories on their origin include genetics, environment, infections, andthe defective failure to process the products of an immune response.

Precise regulation of the expansion and function of T and B cell subsetsis critical for the prevention of autoimmune disorders like SLE, adisease which preferentially affects females (Eisenberg 2003;Cohen-Solal and Diamond 2011). Murine studies have recently uncoveredthe existence of a unique subset of B cells termedage/autoimmunity-associated B cells (ABCs) that preferentially expandsin females with age. In addition to classical B cell markers like B220and CD19, ABCs can also express markers such as CD11c and CD11b and areknown to require T-bet for their generation. Formation of ABCs ispromoted by TLR7/9 engagement and cytokines like IFNγ and IL-21. ABCsincrease prematurely in murine models of lupus and can produceanti-chromatin antibodies. B cells with features similar to ABCs havealso been detected in human autoimmune disorders including SLE. ABCscharacteristically express T-bet and their generation depends on thistranscription factor, hence, these cells are also known as CD11c+Tbet+ Bcells (Naradikian et al. 2016a; Naradikian et al. 2016; Rubtsova et al.2017). The molecular pathways that promote the expansion and pathogenicfunction of ABCs in autoimmune settings are largely unknown.

Multiple lines of evidence have implicated members of the InterferonRegulatory Factor (IRF) family of transcription factors in autoimmunityand lupus development. Amongst the nine IRF family members, IRF4 plays afundamental and multifaceted role in the activation of both T and Bcells (Rogatsky et al. 2014). In addition to IRF4, studies havedemonstrated strong associations between IRF5 variants and humanautoimmune disorders, particularly SLE (Cham et al. 2012; Lazzari andJefferies 2014). It is also unknown whether IRF4 and IRF 5 play a rolein the function of ABCs.

The SWEF family is a small family of proteins comprised of SWAP-70 andits homolog, DEF6 (Gupta et al. 2003; Tanaka et al. 2003; Ripich et al2003). In addition to regulating T and B cell function by regulating theactivity of the transcription factor IRF4 (Biswas et al. 2012; Manni etal. 2015), these proteins also control the cytoskeletal dynamics of Tand B cells by regulating the activation of Rho GTPases (Biswas et al.2010). The SWEF proteins play an important immunoregulatory role in vivoas evidenced by the finding that the simultaneous lack of DEF6 andSWAP-70 (in Doubleknockout=DKO mice) leads to the development of SLE inC57BL/6 mice, which, similarly to human SLE, preferentially affectsfemales (Biswas et al. 2012). The development of autoimmunity in DKOmice is associated with dysregulation of both T and B cell compartmentsincluding expansion of T_(FH) cells, increased IL-21 production, andenhanced formation of germinal centers, and plasma cells (Biswas et al.2012). In further support of a role for this family of proteins inautoimmunity, the DEF6 locus has recently been identified as a geneticrisk factor for human SLE (Sun et al. 2016).

There are currently very few methods of predicting whom will developlupus or other autoimmune diseases and lymphoproliferative disorders,and there are no current therapies for treating SLE other than treatmentof the symptoms, and no preventative therapy. Thus, there is a need inthe art for new treatments and diagnostics for SLE, and other autoimmuneand lymphoproliferative diseases.

SUMMARY OF THE INVENTION

The current invention is based on the discovery that age-associated Bcells (“ABCs”) expand and become pathogenic (“pathogenic ABCs) uponinteraction of certain proteins. In particular, the aberrant expansionof ABCs depends on the transcription factor, interferon regulatoryfactor 5 (“IRF5”). Additionally, the aberrant expansion of ABCs isabolished or decreased by two proteins in the SWEF family, SWAP-70 andDEF6.

One embodiment of the current invention is a method of abolishing ordecreasing pathogenic ABCs in a subject in need thereof by administeringan effective amount of an agent that agonizes, activates or increasesthe expression and/or activity of SWAP-70 and DEF6.

A further embodiment of the current invention is a method of preventingand/or treating an autoimmune or lymphoproliferative disease byabolishing or decreasing pathogenic ABCs in a subject in need thereof byadministering an effective amount of an agent that agonizes, activatesor increases the expression and/or activity of SWAP-70 and DEF6.

Another embodiment of the current invention is a method of abolishing ordecreasing pathogenic ABCs in a subject in need thereof by administeringan effective amount of an agent that antagonizes, inhibits or reducesthe expression and/or activity of IRF5.

A further embodiment of the current invention is a method of preventingand/or treating an autoimmune or lymphoproliferative disease byabolishing or decreasing pathogenic ABCs in a subject in need thereof byadministering an effective amount of an agent that antagonizes, inhibitsor decreases the expression and/or activity of IRF5.

Another embodiment of the current invention is a method of abolishing ordecreasing pathogenic ABCs in a subject in need thereof by administeringan effective amount of an agent that antagonizes, inhibits or reducesthe expression of certain genes that are upregulated in pathogenic ABCs.

A further embodiment of the current invention is a method of preventingand/or treating an autoimmune or lymphoproliferative disease byabolishing or decreasing pathogenic ABCs in a subject in need thereof byadministering an effective amount of an agent that antagonizes, inhibitsor decreases the expression of certain genes that are upregulated inpathogenic ABCs.

A further embodiment of the current invention is a method of abolishingor decreasing pathogenic ABCs in a subject in need thereof byadministering an effective amount of an agent that agonizes, activatesor increases the number or expression of certain genes that aredownregulated in pathogenic ABCs.

Yet a further embodiment of the current invention is a method ofpreventing and/or treating an autoimmune or lymphoproliferative diseaseby abolishing or decreasing pathogenic ABCs in a subject in need thereofby administering an effective amount of an agent that agonizes,activates or increases the number or expression of certain genes thatare downregulated in pathogenic ABCs.

A further embodiment of the invention is a method of preventing and/ortreating an autoimmune disease in a subject in need thereof byadministering an effective amount of an agent that antagonizes orinhibits the ability of pathogenic ABCs to differentiate into plasmacells.

In some embodiments, the autoimmune disease is systemic lupuserythematosus, rheumatoid arthritis, type 1 diabetes, multiplesclerosis, myasthenia gravis, Graves disease, pernicious anemia,scleroderma, psoriasis, inflammatory bowel diseases, Hashimoto'sdisease, Addison's disease, and Sjögren's syndrome. In some embodiments,the lymphoproliferative disease is Hodgkin's lymphoma or non-Hodgkin'slymphoma.

A further embodiment of the current invention is a method of detectingpathogenic ABCs. The detection of pathogenic ABCs makes it possible todiagnose that a subject has an autoimmune or lymphoproliferative diseaseor predict that a subject will develop an autoimmune orlymphoproliferative disease. A method for detecting pathogenic ABCscomprises obtaining a sample from a subject and detecting the presenceof pathogenic ABCs, wherein the pathogenic ABCs comprise B cells thatexpress CD11c, as well as additional markers such as B220, CD86, MHCIIand IgM, but downregulate CD5. The presence of the pathogenic ABCs canalso be detected by detecting the expression of one or more genes ascompared to a reference value of the expression of the same genes inABCs that are not pathogenic or the same genes in other non-pathogenic Bcells, such as follicular B cells.

The expression of the genes from the pathogenic ABCs from a subject witha suspected autoimmune or lymphoproliferative disease can be compared toa reference value of the expression of the same genes in non-pathogenicABCs or other B cells from a healthy donor or control. The levels ofexpressed genes may be measured as absolute or relative. Absolutequantitation measure concentrations of specific RNA and requires acalibration curve. Relative quantification measures fold changedifferences of specific RNA in comparison to housekeeping genes.Relative quantification is usually adequate to investigate physiologicalchanges in gene expression levels.

Once the pathogenic ABCs are detected, a further embodiment of theinvention is abolishing or decreasing the pathogenic ABCs, by themethods set forth herein.

A further embodiment of the current invention is a method of evaluatingor monitoring subjects with an autoimmune or lymphoproliferative diseasefor their response to treatment by the detection of pathogenic ABCs. Amethod for detecting pathogenic ABCs comprises obtaining a sample from asubject, isolating B cells and detecting the presence of pathogenicABCs, wherein the pathogenic ABCs comprise B cells that express cellmarkers CD11c, and additional markers such as B220, CD86, MHC11 and IgM,but downregulate CD5. The presence of the pathogenic ABCs can also bedetected by detecting the expression of one or more genes, as comparedto the expression of the same genes in ABCs that are not pathogenic orthe same genes in other B cells. In this method, the expression of thegenes from the cells of the subject are compared before and aftertreatment.

The present invention also provides for methods and tools for drugdesign, testing of agents, and tools for basic research into the causesand etiology of pathogenic ABCs, and autoimmune or lymphoproliferativedisease.

Thus, a further embodiment of the present invention is a method and/orassay for identifying a test agent for abolishing or decreasingpathogenic ABCs comprising contacting or incubating a test agent with anucleotide comprising the gene for SWAP-70 or DEF6 which expresses ameasurable phenotype, and measuring the phenotype before and aftercontact or incubation with the test agent, wherein if the expression ofthe measurable phenotype is increased after the contact or incubationwith the test agent, the test agent is identified as an agent that canabolish or decrease pathogenic ABCs.

The measurable phenotype can be one that is native to the gene or onethat is artificially linked, such as a reporter gene.

A further embodiment of the present invention is a method and/or assayfor identifying a test agent for abolishing or decreasing pathogenicABCs, comprising transforming a host cell with a gene constructcomprising the gene for SWAP-70 or DEF6, detecting the expression of thegene in the host cell, contacting the test agent with the host cell, anddetecting the expression of the gene from the host cell after contactwith the test agent or compound, wherein if the expression of the geneis increased after contact with the test agent or compound, the testagent is identified as an agent that can abolish or decrease pathogenicABCs.

One embodiment is a method and/or assay for identifying a test agent forabolishing or decreasing pathogenic ABCs, comprising contacting orincubating the test agent with IRF5, and detecting the presence of acomplex between the test agent, wherein if a complex between the testagent and IRF5 is detected, the test agent is identified as an agentthat can abolish or decrease pathogenic ABCs.

Another embodiment of the present invention is a method and/or assay foridentifying a test agent for abolishing or decreasing pathogenic ABCs,comprising contacting or incubating the test agent with IRF5 and a knownantibody of IRF5, and detecting the presence and quantity of unboundantibody, wherein the presence of the unbound antibody indicates thatthe test agent is binding to IRF5 and the test agent is identified as anagent that can abolish or decrease pathogenic ABCs.

A further embodiment of the present invention is a method and/or assayfor identifying a test agent for abolishing or decreasing pathogenicABCs comprising contacting or incubating a test agent to a nucleotidecomprising the gene for IRF5, and determining if the test agent binds tothe gene, wherein if the test agent binds to the nucleotide, the testagent is identified as an agent that can abolish or decrease pathogenicABCs.

A further embodiment of the present invention is a method and/or assayfor identifying a test agent for abolishing or decreasing pathogenicABCs comprising contacting or incubating a test agent with a nucleotidecomprising the gene for IRF5 which expresses a measurable phenotype, andmeasuring the phenotype before and after contact or incubation with thetest agent, wherein if the expression of the measurable phenotype isdecreased after the contact or incubation with the test agent, the testagent is identified as an agent that can abolish or decrease pathogenicABCs.

The measurable phenotype can be one that is native to the gene or onethat is artificially linked, such as a reporter gene.

One embodiment of the present invention is a method and/or assay foridentifying a test agent for abolishing or decreasing pathogenic ABCs,comprising transforming a host cell with a gene construct comprising thegene for IRF5, detecting the expression of the gene in the host cell,contacting the test agent with the host cell, and detecting theexpression of the gene from the host cell after contact with the testagent or compound, wherein if the expression of the gene is reduced ordecreased after contact with the test agent or compound, the test agentis identified as an agent that can abolish or decrease pathogenic ABCs.

A further embodiment of the present invention is a method and/or assayfor identifying a test agent for abolishing or decreasing pathogenicABCs comprising contacting or incubating a test agent to a nucleotidecomprising a gene that is upregulated in pathogenic ABCs, anddetermining if the test agent binds to the gene, wherein if the testagent binds to the nucleotide, the test agent is identified as an agentthat can abolish or decrease pathogenic ABCs.

A further embodiment of the present invention is a method and/or assayfor identifying a test agent for abolishing or decreasing pathogenicABCs comprising contacting or incubating a test agent with a nucleotidecomprising a gene that is upregulated in pathogenic ABCs which expressesa measurable phenotype, and measuring the phenotype before and aftercontact or incubation with the test agent, wherein if the expression ofthe measurable phenotype is decreased after the contact or incubationwith the test agent, the test agent is identified as an agent that canabolish or decrease pathogenic ABCs.

The measurable phenotype can be one that is native to the gene or onethat is artificially linked, such as a reporter gene.

A further embodiment of the present invention is a method and/or assayfor identifying a test agent for abolishing or decreasing pathogenicABCs, comprising transforming a host cell with a gene constructcomprising a gene that is upregulated in pathogenic ABCs, detecting theexpression of the gene in the host cell, contacting the test agent withthe host cell, and detecting the expression of the gene from the hostcell after contact with the test agent or compound, wherein if theexpression of the gene is reduced or decreased after contact with thetest agent or compound, the test agent is identified as an agent thatcan abolish or decrease pathogenic ABCs.

A further embodiment of the present invention is a method and/or assayfor identifying a test agent for abolishing or decreasing pathogenicABCs comprising contacting or incubating a test agent with a nucleotidecomprising a gene that is downregulated in pathogenic ABCs whichexpresses a measurable phenotype, and measuring the phenotype before andafter contact or incubation with the test agent, wherein if theexpression of the measurable phenotype is increased after the contact orincubation with the test agent, the test agent is identified as an agentthat can abolish or decrease pathogenic ABCs.

The measurable phenotype can be one that is native to the gene or onethat is artificially linked, such as a reporter gene.

A further embodiment of the present invention is a method and/or assayfor identifying a test agent for abolishing or decreasing pathogenicABCs, comprising transforming a host cell with a gene constructcomprising a gene that is downregulated in pathogenic ABCs, detectingthe expression of the gene in the host cell, contacting the test agentwith the host cell, and detecting the expression of the gene from thehost cell after contact with the test agent or compound, wherein if theexpression of the gene is increased after contact with the test agent orcompound, the test agent is identified as an agent that can abolish ordecrease pathogenic ABCs.

Any test agent identified by these methods and assays would be useful inpreventing and/or treating an autoimmune or lymphoproliferative disease.

The present invention also includes kits.

BRIEF DESCRIPTION OF THE FIGURES

For the purpose of illustrating the invention, there are depicted indrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

Certain abbreviations are used in the Figures and Brief Description ofthe Figures including: WT—wild type; FACS—flow cytometry analysis; andFoB cells—follicular B cells.

FIG. 1 shows the result showing the spontaneous expansion of ABCs in DKOmice. FIG. 1A is representative flow cytometric analysis (FACS) plotsand graphs of ABC cells in the spleens from aging (greater than 23weeks-old) wild type (WT) or DKO female mice. Graphs show frequenciesand numbers of individual mice and mean value of 4-8 independentexperiments (n=3-16). **: p≤0.01, ****: p≤0.0001. (two-tailed Studentt-test). FIG. 1B is representative FACS plots and graphs for CD11b andCD11c expression after gating on B220+CD19+ cells in skin draining lymphnodes of WT, DKO, Def6ko (Def6^(tr/tr)), and SWAP70ko (Swap-70^(−/−))mice (18-24 weeks old). Graphs show frequencies and numbers of cells inindividual mice and mean value of 3 independent experiments (n=3-5). **:p≤0.01. (One-way ANOVA). FIG. 1C is representative FACS plots and graphsfor CD11b and CD11c expression after gating on B220+CD19+ cells in thespleens of 10 weeks old WT and DKO female mice. Graphs show frequenciesand numbers of individual mice and mean value of 2 independentexperiments (n=4-5). **: p≤0.01; ***: p≤0.001. (two-tailed Student-ttest). FIG. 1D is representative FACS plots and graphs for CD11c andCD11b expression gated on B220+CD19+ cells in the spleens of WT, DKO,Def6ko and Swap70ko female mice (18-24 weeks old). Graphs showfrequencies and numbers for individual mice and mean values from 3independent experiments (n=3-5). ****: p≤0.0001 (One-way ANOVA). FIG. 1Eare histograms that show relative expression (percentage) of theindicated marker on CD11c+CD11b+B220+CD19+ and CD11c−CD11b−B220+CD19+cells in the spleens of DKO female mice (greater than 18 weeks old).Data are representative of least 2 independent experiments (n=4-11).FIG. 1F is representative FACS plots and graphs for IgG1 and IgG2cexpression on B220+CD19+CD11c+CD11b+ cells. Graphs show frequencies andnumbers for individual mice and mean values from 16 independentexperiments (n=17-30). **: p≤0.01, ***: p≤0.001, ****: p≤0.0001.(two-tailed Student-t test). FIG. 1G are graphs of the levels ofanti-dsDNAIgG2c, anti-nRNP, and anti-Cardiolipin IgG antibodies in thesupernatants of sorted ABC (CD11c+CD11b+B220+CD19+) and FoB(CD11c−CD11b−CD23+B220+CD19+) B cells stimulated in vitro±1 μg/mlimiquimod for 7 days as measured by ELISA (representative of 4independent experiments). Mean±SEM is shown. ***: p≤0.001 (One-wayANOVA).

FIG. 2 shows the results that IL-21 regulates the generation of DKO ABCsin vitro and in vivo. FIG. 2A is representative FACS plots and graph ofthe generation of ABCs (CD11c+T-bet+B220+) from cultures of CD23+ Bcells purified from WT and DKO female mice (8-10 weeks of age)stimulated with αIgM and αCD40, alone or with IL-21 or imiquimod for 3days. Graph shows frequencies of cells in mice as combined results of 5independent experiments. Mean±SEM is shown. ****: p≤0.0001. (One-wayANOVA). FIG. 2B is representative FACs plots and graph of generation ofABCs (CD11c+CD11b+B220+) B cells from cultures of CD23+ B cells purifiedfrom WT and DKO female mice (8-10 weeks of age) stimulated with αIgM andαCD40 (5 μg/ml), alone or with IL-21 or imiquimod. Graph showsfrequencies of cells in mice as combined results of 5 independentexperiments. ***: p≤0.001. (One-way ANOVA). FIG. 2C is representativeFACS plots and graphs of ABCs in the spleens from aging (greater than 24weeks-old) wild type, DKO, or IL-21ko DKO female mice for CD11c andCD11b expression gated on B220+ cells. Graphs show the frequencies andnumbers of CD11c+CD11b+ cells of individual mice gated as indicated aswell as the mean value of at least 4 independent experiments (n=5-8)***: p≤0.001, ****: p≤0.0001 (One-way ANOVA). FIG. 2D showsrepresentative FACS plots and graphs of ABCs in the spleens from aging(greater than 24 weeks-old) wild type, DKO, or SAPko DKO female mice forCD11c and CD11b (left panels) expression gated on B220+ cells. Graphsshow the frequencies and numbers of CD11c+CD11b+ cells of individualmice gated as indicated as well as the mean value of at least 4independent experiments (n=4-7). ****: p≤0.0001. FIG. 2E show graphs ofamount of IgG anti-ds DNA from serum from the indicated mice as assayedby ELISA. Graphs show data of individual mice and mean value of 4independent experiments (n=6-8). ****: p≤0.0001. FIG. 2F are graphsshowing the quantification (both in percentages and absolute numbers) ofT_(FH) (CD4+CXCR5+PD1+Foxp3−), germinal center (GC) B cells(B220+FAS+GL-7+), and B220^(int)CD138+ plasma cells (PC) in spleens ofWT, DKO, IL-21ko DKO or SAPko DKO female mice (>24 weeks old). Graphsshow percentages and numbers of specific cells types in individual miceper genotype (n=4-8). *: p≤0.05; **: p≤0.01; ***: p≤0.001. ****:p≤0.0001. (One-way ANOVA).

FIG. 3 shows the results showing differentially expressed genes inpathogenic ABCs. FIG. 3A shows the hierarchical clustering oflog-transformed counts per million (cpm) for differentially expressedgenes identified by RNAseq analysis from FACS sorted ABC(B220⁺CD19⁺CD11c⁺CD11b⁺) from DKO female mice or FoB(B220⁺CD19⁺CD11c⁻CD11b⁻CD23⁺) cells from WT and DKO female mice (greaterthan 20 weeks old) (n=2 WT, 3 DKO/group). FIG. 3B is a volcano plot ofdifferentially expressed genes in FoB wild type and FoB DKO mice. Colorsindicate differentially expressed genes (P value<0.01, Fold change>2)belonging to selected GSEA Hallmark pathways as indicated. FIG. 3C showsthe results of flow cytometry analysis showing the proliferation ofCD11c−T-bet−B220+ cells in the spleens of WT and DKO female mice (>23weeks old, left panel) or of CD11c−T25 bet−B220+ cells andCD11c+T-bet+B220+ (ABCs) in the spleens of DKO female mice (>23 weeksold) as assessed by Ki67 staining and flow cytometry. Shown is arepresentative histogram of 4 and 5 independent experiments,respectively (n=5 and 6 mice/group, respectively). FIG. 3D are graphsshowing the apoptotic rate as measured by caspase 3 cleavage inCD11c−T-bet−B220+ B cells in WT and DKO mice or in CD11c−T-bet− B cellsand CD11c+T-bet+ (ABC) B cells in DKO mice. Shown is a representativehistogram. All data are representative of 3 independent experiments.(n=4 mice). FIG. 3E is a representative histogram of 5 independentexperiments (n=5) showing the results of the proliferation ofCD11c−T-bet−B220+ cells and ABCs (CD11c+T-bet+B220+) assessed byevaluating dilution of cell trace violet by flow cytometry. FIG. 3F aregraphs of cell viability of CD23+ B cells purified from WT and DKOfemale mice (6-9 weeks old) and stimulated with αIgM and αCD40, alone orwith IL-21 or imiquimod for 3 or 5 days assessed by staining withCaspGlow orcpropidium iodide as indicated. FIG. 3G is a volcano plotcomparing gene expression in FoB (CD11c−CD11b−CD23+B220+CD19+) and ABC(CD11c+CD11b+B220+CD19+) cells sorted from DKO female mice (>20 weeksold). Colors indicate differentially expressed genes (P value<0.01, Foldchange>2, DKOFoB/DKO ABC) belonging to selected GSEA pathways asindicated. FIG. 3H shows hierarchical clustering of log-transform countsper million (cpm) for genes that belong to the GO_inflammatory_responsegene set (MsigDB) identified by RNA-Seq analysis of RNA from FACS sortedFoB (B220+CD19+CD11c−CD11b−CD23+) and ABC (B220+CD19+CD11c+CD11b+) cellsfrom DKO female mice (n=3). FIG. 3I are graphs that show the results ofqPCR analysis of the expression of Ccl5, Ifnγ, and Cxcl10 mRNA in sortedFoB (B220+CD19+CD11c−CD11b−CD23+) cells from WT and DKO female mice andABC (B220+CD19+CD11c+CD11b+) cells from DKO female mice as indicated.The data were normalized relative to ppia mRNA expression. Data arerepresentative of 2 (Ccl5) or 3 (Ifnγ and Cxcl10) independentexperiments. FIG. 3J are graphs of qPCR analysis of the expression ofthe indicated mRNA (gene) in FACS sorted FoB(B220+CD19+CD11c−CD11b−CD23+) cells from WT and DKO female mice and ABC(B220+CD19+CD11c+CD11b+) cells from DKO female mice. The data werenormalized relative to ppia mRNA expression. FIG. 3K shows selected GSEApathways analysis of DKO FoB compared to DKO ABC cells.

FIG. 4 shows that the chromatin landscape of DKO ABCs is enriched in IRFand AP-1/BATF motifs. FIG. 4A shows the normalized ATAC-seq tag densitydistributions for 4 kb window centered at the summit of ABC-specificpeaks (n=3,666, top panel) and average distribution of ATAC-seqnormalized tag densities (bottom). (n=2/group). FIG. 4B arerepresentative UCSC Genome Browser tracks displaying ATAC-seq normalizedtag densities at representative genomic regions Cxcl10 cluster, Il6, andIFNγ genes. Highlighted are ABC-specific ATAC-seq peaks. FIG. 4C isgraph of motif density distribution relative to the peak summit for IRF,T-bet and POU2F2 motifs in ABC-specific ATAC-seq peaks. FIG. 4D is agraph of normalized tag counts for ABC and FOB expression (RNAseq) oftotal ABC-specific peaks associated genes (n=2,482) from threeindependent experiments (n=3) normalized tag counts.

FIG. 5 shows the results showing WT and DKO ABCs exhibit a distinctivetranscriptional and chromatin profile. FIG. 5A is a graph of theexpression of selected transcription factors transcripts in WT and DKOABC cells as identified by RNA-seq analysis. FIG. 5B shows thehierarchical clustering of log-transformed counts per million (cpm) fordifferentially expressed genes identified by RNAseq analysis of RNA fromFACS sorted ABC (B220+CD19+CD11c+CD11b+) cells from WT and DKO femalemice (>20 weeks old). (n=2/group). FIG. 5C shows violin plot analysis ofall genes, Ig genes and ARCHS4 Macrophage genes in WT and DKO. FIG. 5Dare graphs of the results of qPCR analysis of the expression ofrepresentative genes in sorted ABC (B220+CD19+CD11c+CD11b+) cells fromWT and DKO female mice as indicated. The data were normalized relativeto ppia mRNA expression. Data are representative of 2 independentexperiments. ns: not significant, *: p≤0.05, **: p≤0.01 ***: p≤0.001.FIG. 5E are graphs of qPCR expression analysis of the indicated genes inFACS sorted ABC (B220+CD19+CD11c+CD11b+) cells from WT and DKO femalemice (>24 weeks). The data were normalized relative to ppia mRNAexpression. FIG. 5F show normalized ATAC-seq tag density distributionsfor 4 kb window centered at the summit of WT-specific (right, n=27,483)or DKO-specific (left, n=1583) peaks and average distribution ofATAC-seq normalized tag densities (bottom). (n=2/group). Histograms showexpression (RNAseq) of total ABC-specific peaks associated genes in WTand DKO from three independent experiments (n=2-3). The graphs show thequantitated results. FIG. 5G are graphs of functionally enriched GeneOntology (GO) categories of WT-specific and DKO-specific peaks ofATAC-seq.

FIG. 6 shows the results that IRF5 regulates the IL-21-mediatedformation of DKO ABCs. FIG. 6A is representative FACS plots of purifiedwild type, DKO and CD21Cre IRF5^(fl/−)DKO (8-10 weeks of age) CD23+Bcells were treated with a combination of αCD40, αIgM, IL-21 and/orimiquimod. CD11c+Tbet+Bcells in each culture condition were assayed byFACS at day 3. Representative FACS plot of 6 independent experiments isshown. FIG. 6B shows the graphical results of percentage of ABCs fromthe FAC analysis in FIG. 6A (n=6). Mean±SEM is shown. **: p≤0.01, ***:p≤0.001. (One-way ANOVA). FIG. 6C shows graphs of the analysis of theexpression and production of IL-6 in cultures of cells stimulated±IL-21as assessed by qPCR and ELISA. qPCR data were normalized relative toppia mRNA expression. Data are representative of 3 independentexperiments. Mean±SEM is shown. ns: not significant, ***: p≤0.001, ****:p≤0.0001. (One-way ANOVA). FIG. 6D shows graphs of the analysis of theexpression and production of Cxcl10 in cultures of cells stimulated withand without IL-21 as assessed by qPCR (left panel) and ELISA (rightpanel). qPCR data were normalized relative to ppia mRNA expression. Dataare representative of 3 independent experiments. Mean±SEM is shown. ns:not significant, ***: p≤0.001, ****: p≤0.0001. (One-way ANOVA). FIG. 6Eshows graphs of amount of IgG1 and IgG2c in the supernatants of cellsstimulated±IL-21 7 days as analyzed by ELISA. Data are representative of3 independent experiments. Mean±SEM is shown. ns: not significant, ****:p≤0.0001. (One-way ANOVA). FIG. 6F shows the results of qPCR analysis ofthe expression of Jun in cultures of cells stimulated±IL-21. Data werenormalized relative to ppia mRNA expression. Data are representative of2 independent experiments. Mean±SEM is shown. **: p≤0.01 ***: p≤0.001;****: p≤0.0001. FIG. 6G are graphs of the results of ChIP assaysperformed with an IRF5 antibody on cells stimulated with IL-21 for 2days. Immunoprecipitated DNA was analyzed by qPCR using primers withinthe ABC-specific ATAC-seq peaks at the CXCL10 cluster (Cl), IgG2c, Jun,and the IL-6 TSS. Data are representative of 4 (IL-6 TSS and CXCL10 Cl)or 2 (IgG2c and Jun) independent experiments. Mean±SEM is shown. ***:p≤0.001; ****: p≤0.0001. (One-way ANOVA). FIG. 6H is a Western blot ofnuclear extracts prepared from CD23+ B cells purified from WT, DKO andCD21Cre IRF5^(fl/−) DKO female mice (8-10 weeks of age) stimulated withαIgM (5 μg/ml), αCD40 (5 μg/ml), IL-21 (50 ng/ml) or imiquimod (1 μg/ml)for 3 days. Extracts were analyzed by Western blotting with pSTAT3,STAT3, IRF5, and HDAC1 antibodies. Data are representative of 2independent experiments. FIG. 6I are graphs of the results of a ChIPassay performed with a T-bet antibody. Data are representative of 4(IL-6 TSS and CXCL10 Cl) or 2 (IgG2c and Jun) independent experiments.Mean±SEM is shown. *: p≤0.05, **: p≤0.01 ***: p≤0.001; ****: p≤0.0001.FIG. 6J show Western Blots of nuclear extracts prepared from cellsstimulated with and without IL-21 for 2 days and subjected to ONP assaywith a biotinylated oligonucleotide from the CXCL10 Cl. Precipitatedproteins were analyzed by Western blotting with an IRF5 and T-betantibody as indicated. Data are representative of 2 independentexperiments. FIG. 6K shows Western Blots of nuclear extracts from 293Tcells transiently transfected as indicated and subjected to ONP assaywith a biotinylated oligonucleotide from the CXCL10 Cl or IL-6 TSS.Precipitated proteins were analyzed by Western blotting with an IRF5 orT-bet antibody. Data are representative of 2 independent experiments.FIG. 6L is a Western blot of IRF5/SWEF proteins coimmunoprecipitationfrom nuclear extracts from cells stimulated with or without IL-21 for 2days. Immunoprecipitation was performed with an IRF5 antibody and probedwith a DEF6, SWAP-70 or IRF5 antibody as indicated. Data arerepresentative of 2 independent experiments. FIG. 6M is a Western blotof nuclear extracts of 293T cells transiently transfected with variousconstructs as indicated. Immunoprecipitations were performed using ananti-HA antibody. Immunoprecipitates were analyzed by Western blottingusing HA antibodies. Data are representative of 2 independentexperiments with similar results. FIG. 6N is a Western blot of nuclearextracts of 293T cells subjected to ONP assay with a biotinylatedoligonucleotide from the IL-6 TSS. Precipitated proteins were analyzedby Western blotting with an IRF5 antibody. Data are representative of 2independent experiments. FIG. 6O is a Western blot of 293T cellstransiently transfected with various constructs as indicated.Immunoprecipitations were performed using an anti-HA antibody.Immunoprecipitates were analyzed by Western blotting using an anti-FLAG,T-bet or HA antibodies. Data are representative of 2 independentexperiments with similar results.

FIG. 7 shows the results that monoallelic deletion of IRF5 abolishesaccumulation of ABCs and lupus development in DKO mice. FIG. 7A showsrepresentative FACS plots of CD11c and CD11b expression in CD11c+CD11b+B cells in the spleens of WT, IRF5^(fl/fl) DKO, IRF5^(fl/−) DKO andCD21Cre IRF5^(fl/−) DKO female mice (>20 weeks-old). FIG. 7B are graphsof the percentage and absolute numbers of ABCs (CD11c+CD11b+B220+) inmice of the indicated genotypes. (n=5-10 mice). ****: p≤0.0001. (One-wayANOVA). FIG. 7C are graphs of percentages and absolute numbers ofB220+CD19+CD11c+Tbet+ B cells in the spleens of WT, IRF5^(fl/fl) DKO,IRF5^(fl/−) DKO, CD11cCre IRF5^(fl/−) DKO and CD21Cre IRF5^(fl/−) DKOfemale mice (>20 weeks-old). Graphs show percentages and numbers inindividual mice (n=5-7). ***: p≤0.001; ****: p≤0.0001. (One-way ANOVA).FIG. 7D are graphs of percentages and absolute numbers ofB220+CD19+CD21−CD23−CD11c+CD11b+ B cells in the spleens of WT,IRF5^(fl/fl) DKO, IRF5^(fl/−) DKO, CD11cCre IRF5^(fl/−) DKO and CD21CreIRF5^(fl/−) DKO female mice (>20 weeks-old). Graphs show percentages andnumbers in individual mice (n=5-7). ***: p≤0.001; ****: p≤0.0001.(One-Way ANOVA). FIG. 7E are graph shows percentages and absolutenumbers of total splenocytes in WT, IRF5^(fl/fl) DKO, IRF5^(fl/−) DKO,CD11cCre IRF5^(fl/−) DKO and CD21Cre IRF5^(fl/−) DKO mice (>20 weeks ofage) (n=5-10 mice). *: p≤0.05; ***: p≤0.001, ****: p≤0.0001. (One-wayANOVA). FIG. 7F are graphs quantifying flow cytometric analysis ofT_(FH) (CD4+CXCR5+PD1+Foxp3−), germinal center (GC) B cells(B220+FAS+GL-7+), and plasma cells (PC) (B220^(int)CD138+) in spleens ofWT, IRF5^(fl/fl) DKO, IRF5^(fl/−) DKO, CD11cCre IRF5^(fl/−) DKO andCD21Cre IRF5^(fl/−) DKO mice (>20 weeks). Graphs show percentages andnumbers of specific cells types in individual mice (n=5-10). **: p≤0.01;***: p≤0.001; ****: p≤0.0001. (One-way ANOVA). FIG. 7G are graphsquantifying flow cytometric analysis of Treg (CD4+Foxp3+), activatedTreg (CD4+Foxp3+CD44+), and activated T cells (CD4+Foxp3−CD44+) inspleens of WT, IRF5^(fl/fl) DKO, IRF5^(fl/−) DKO, CD11cCre IRF5^(fl/−)DKO and CD21Cre IRF5^(fl/−) DKO mice ((>20 weeks). Graphs showfrequencies and numbers of individual mice and mean value of 10independent experiments (n=5-10). **: p≤0.01; ***: p≤0.001; ****:p≤0.0001. (One-way ANOVA). FIG. 7H show graphs of the intensity score ofstained cells to determine anti-nuclear antibodies (ANAs) in sera(1:200) from WT, IRF5^(fl/fl) DKO, IRF5^(fl/−) DKO, CD11cCre IRF5^(fl/−)DKO and CD21Cre IRF5^(fl/−) DKO mice (>20 weeks old). Graph shows scoreof individual mice and mean value of 10 independent experiments(n=7-12). *: p≤0.05, **: p≤0.01 ***: p≤0.001 (Mann-Whitney test). FIG.7I are graphs of the levels of anti-dsDNA IgG, IgG1, or IgG2c antibodiesin the sera of WT, IRF5^(fl/fl) DKO, IRF5^(fl/−) DKO, CD11cCreIRF5^(fl/−) DKO and CD21Cre IRF5^(fl/−) DKO female mice (>20 weeks old)analyzed by ELISA (n=4-11). *: p≤0.05; ***: p≤0.001; ****: p≤0.0001.(One-way ANOVA). FIG. 7J are graphs of the levels of anti-ssDNA,cardiolipin and nRNP IgG antibodies in the sera of WT, IRF5^(fl/fl) DKO,IRF5^(fl/−) DKO, CD11cCre IRF5^(fl/−) DKO and CD21Cre IRF5^(fl/−) DKOfemale mice (>20 weeks old) analyzed by ELISA (n=4-19). *: p≤0.05; **:p≤0.01, ***: p≤0.001; ****: p≤0.0001. (One-way ANOVA). FIG. 7K show agraph of the glomerulonephritis score of WT, DKO and IRF5ko DKO mice(which include CD11cCre IRF5fl/− DKO and CD21Cre IRF5fl/− DKO mice)(n=3-6 mice/group). The graphs are quantification of PAS staining of 3independent experiments. FIG. 7L is a graph showing the MFIquantification of IgG deposition from 3 independent experiments. (n=3-6mice/group).

FIG. 8 shows that a subset of CD11c+ DKO B cells upregulates PC markers.FIG. 8A is a representative FACS plot for CD11c and CD11b expressiongated on CD19 Blimp1-yfphi cells from spleens of aging Blimp1-yfp.DKOfemale mice. FIG. 8B are histograms showing the relative expression ofthe indicated marker on CD11c+ PCs (CD11c+ Blimp1hi CD19lo=red), ABCs(CD11c+CD11b+CD19+Blimp1−=blue), and total B cells (CD19+=shaded gray)from aging Blimp1-yfp.DKO female mice.

FIG. 9 shows that DKO ABCs exhibit sex specific differences inautoantibody production. FIG. 9A is a scatter plot showing frequenciesof ABCs of individual mice as indicated. FIG. 9B is a graph of theresults of ABCs sorted from spleens of aging DKO female or DKO male micecultured in vitro±1 μg/ml of Imiquimod, assayed by ELISA on day 7. FIG.9C is a graph of the results of ABCs sorted from spleens of aging DKOmale or Yaa-DKO male mice cultured in vitro±1 μg/ml of Imiquimod,assayed by ELISA on day 7. FIG. 9D show graphs of frequencies of ABCexpressing the indicated markers using flow cytometric analysis of B220+B cells in the spleens of WT and DKO female and male mice and Yaa-DKOmale mice (>20 weeks-old). FIG. 9E is a graph showing the qPCRexpression analysis of IL13Ra1 in FACS-sorted FoB(B220+CD19+CD11c−CD11b−CD23+) cells and ABC (B220+CD19+CD11c+CD11b+)cells from WT and DKO female and male mice and Yaa-DKO male mice (>20weeks old). The data were normalized relative to ppia mRNA expression.

FIG. 10 show the expression of Rho-kinase 1 and 2 in differentpopulations of cells. FIG. 10A shows the indicated cells assayed withROCK2 in vitro kinase assay. FIG. 10B shows the expression of phosphoIRF4 in some of the same cells (plasmablasts) as assessed by Westernblotting with a pIRF4 Antibody. FIG. 10C shows the same cells used inFIG. 10A assayed with a ROCK1 in vitro kinase assay.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the methods of the invention and howto use them. Moreover, it will be appreciated that the same thing can besaid in more than one way. Consequently, alternative language andsynonyms may be used for any one or more of the terms discussed herein,nor is any special significance to be placed upon whether or not a termis elaborated or discussed herein. Synonyms for certain terms areprovided. A recital of one or more synonyms does not exclude the use ofthe other synonyms. The use of examples anywhere in the specification,including examples of any terms discussed herein, is illustrative only,and in no way limits the scope and meaning of the invention or anyexemplified term. Likewise, the invention is not limited to itspreferred embodiments.

The terms “pathogenic age-associated B cells”, and “pathogenic ABCs” isused herein to denote the novel subset of B cells described herein thatpromote disease. The terms “DKO ABCs” and “autoimmune prone ABCs” alsodenote these cells.

The term “subject” as used in this application means an animal with animmune system such as avians and mammals. Thus, the invention can beused in veterinary medicine, e.g., to treat companion animals, farmanimals, laboratory animals in zoological parks, and animals in thewild. The invention is particularly desirable for human medicalapplications.

The term “patient” as used in this application means a human subject. Insome embodiments of the present invention, the “patient” is onesuffering with an autoimmune or lymphoproliferative disease or suspectedof suffering from an autoimmune or lymphoproliferative disease, such assystemic lupus erythematosus or lymphoma.

The term “detection”, “detect”, “detecting” and the like as used hereinmeans as used herein means to discover the presence or existence of.

The terms “diagnosis”, “diagnose”, diagnosing” and the like as usedherein means to determine what physical disease or illness a subject orpatient has, in this case an autoimmune or lymphoproliferative disease.

The terms “identification”, “identify”, “identifying” and the like asused herein means to recognize a disease state or a clinicalmanifestation or severity of a disease state in a subject or patient.The term also is used in relation to test agents and their ability tohave a particular action or efficacy.

The terms “prediction”, “predict”, “predicting” and the like as usedherein means to tell in advance based upon special knowledge.

The term “reference value” as used herein means an amount or a quantityof a particular protein or nucleic acid in a sample from a healthycontrol or healthy donor.

The terms “healthy control”, “healthy donor” and “HD” are usedinterchangeably in this application and are a human subject who is notsuffering from systemic lupus erythematosus or any other autoimmune orlymphoproliferative disease.

The terms “treat”, “treatment”, and the like refer to a means to slowdown, relieve, ameliorate or alleviate at least one of the symptoms ofthe disease, or reverse the disease after its onset.

The terms “prevent”, “prevention”, and the like refer to acting prior toovert disease onset, to prevent the disease from developing or minimizethe extent of the disease or slow its course of development.

The term “agent” as used herein means a substance that produces or iscapable of producing an effect and would include, but is not limited to,chemicals, pharmaceuticals, biologics, small organic molecules,antibodies, nucleic acids, peptides, and proteins.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to cause an improvement in a clinically significantcondition in the subject, or delays or minimizes or mitigates one ormore symptoms associated with the disease, or results in a desiredbeneficial change of physiology in the subject.

The phrase “in need thereof” indicates a subject has an autoimmune orlymphoproliferative disease, is suspected of having an autoimmune orlymphoproliferative disease, or has risk factors for an autoimmune orlymphoproliferative disease.

The terms “expression profile” or “gene expression profile” refers toany description or measurement of one or more of the genes that areexpressed by a cell, tissue, or organism under or in response to aparticular condition. Expression profiles can identify genes that areupregulated, downregulated, or unaffected under particular conditions.Gene expression can be detected at the nucleic acid level or at theprotein level. The expression profiling at the nucleic acid level can beaccomplished using any available technology to measure gene transcriptlevels. For example, the method could employ in situ hybridization,Northern hybridization or hybridization to a nucleic acid microarray,such as an oligonucleotide microarray, or a cDNA microarray.Alternatively, the method could employ reverse transcriptase-polymerasechain reaction (RT-PCR) such as fluorescent dye-based quantitative realtime PCR (TaqMan® PCR). In the Examples section provided below, nucleicacid expression profiles were obtained using Affymetrix GeneChip®oligonucleotide microarrays. The expression profiling at the proteinlevel can be accomplished using any available technology to measureprotein levels, e.g., using peptide-specific capture agent arrays.

The terms “gene”, “gene transcript”, and “transcript” are used somewhatinterchangeable in the application. The term “gene”, also called a“structural gene” means a DNA sequence that codes for or corresponds toa particular sequence of amino acids which comprise all or part of oneor more proteins or enzymes, and may or may not include regulatory DNAsequences, such as promoter sequences, which determine for example theconditions under which the gene is expressed. Some genes, which are notstructural genes, may be transcribed from DNA to RNA, but are nottranslated into an amino acid sequence. Other genes may function asregulators of structural genes or as regulators of DNA transcription.“Transcript” or “gene transcript” is a sequence of RNA produced bytranscription of a particular gene. Thus, the expression of the gene canbe measured via the transcript.

The term “antisense DNA” is the non-coding strand complementary to thecoding strand in double-stranded DNA.

The term “nucleic acid hybridization” refers to anti-parallel hydrogenbonding between two single-stranded nucleic acids, in which A pairs withT (or U if an RNA nucleic acid) and C pairs with G. Nucleic acidmolecules are “hybridizable” to each other when at least one strand ofone nucleic acid molecule can form hydrogen bonds with the complementarybases of another nucleic acid molecule under defined stringencyconditions. Stringency of hybridization is determined, e.g., by (i) thetemperature at which hybridization and/or washing is performed, and (ii)the ionic strength and (iii) concentration of denaturants such asformamide of the hybridization and washing solutions, as well as otherparameters. Hybridization requires that the two strands containsubstantially complementary sequences. Depending on the stringency ofhybridization, however, some degree of mismatches may be tolerated.Under “low stringency” conditions, a greater percentage of mismatchesare tolerable (i.e., will not prevent formation of an anti-parallelhybrid).

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g. a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g. transcription and translation) of the introducedsequence. Vectors include, but are not limited to, plasmids, phages, andviruses.

The term “host cell” means any cell of any organism that is selected,modified, transformed, grown, used or manipulated in any way, for theproduction of a substance by the cell, for example, the expression bythe cell of a gene, a DNA or RNA sequence, a protein or an enzyme. Hostcells can further be used for screening or other assays, as describedherein.

A “polynucleotide” or “nucleotide sequence” is a series of nucleotidebases (also called “nucleotides”) in a nucleic acid, such as DNA andRNA, and means any chain of two or more nucleotides. A nucleotidesequence typically carries genetic information, including theinformation used by cellular machinery to make proteins and enzymes.These terms include double or single stranded genomic and cDNA, RNA, anysynthetic and genetically manipulated polynucleotide, and both sense andanti-sense polynucleotide. This includes single- and double-strandedmolecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as“protein nucleic acids” (PNA) formed by conjugating bases to an aminoacid backbone. This also includes nucleic acids containing modifiedbases, for example thio-uracil, thio-guanine and fluoro-uracil.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The nucleicacids herein may be flanked by natural regulatory (expression control)sequences, or may be associated with heterologous sequences, includingpromoters, internal ribosome entry sites (IRES) and other ribosomebinding site sequences, enhancers, response elements, suppressors,signal sequences, polyadenylation sequences, introns, 5′- and3′-non-coding regions, and the like. The term encompasses nucleic acidscontaining known nucleotide analogs or modified backbone residues orlinkages, which are synthetic, naturally occurring, and non-naturallyoccurring, which have similar binding properties as the referencenucleic acid, and which are metabolized in a manner similar to thereference nucleotides. The nucleic acids may also be modified by manymeans known in the art. Non-limiting examples of such modificationsinclude methylation, “caps”, substitution of one or more of thenaturally occurring nucleotides with an analog, and internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoroamidates, andcarbamates) and with charged linkages (e.g., phosphorothioates, andphosphorodithioates). Polynucleotides may contain one or more additionalcovalently linked moieties, such as, for example, proteins (e.g.,nucleases, toxins, antibodies, signal peptides, and poly-L-lysine),intercalators (e.g., acridine, and psoralen), chelators (e.g., metals,radioactive metals, iron, and oxidative metals), and alkylators. Thepolynucleotides may be derivatized by formation of a methyl or ethylphosphotriester or an alkyl phosphoramidate linkage. Modifications ofthe ribose-phosphate backbone may be done to facilitate the addition oflabels, or to increase the stability and half-life of such molecules inphysiological environments. Nucleic acid analogs can find use in themethods of the invention as well as mixtures of naturally occurringnucleic acids and analogs. Furthermore, the polynucleotides herein mayalso be modified with a label capable of providing a detectable signal,either directly or indirectly. Exemplary labels include radioisotopes,fluorescent molecules, and biotin.

The term “polypeptide” as used herein means a compound of two or moreamino acids linked by a peptide bond. “Polypeptide” is used hereininterchangeably with the term “protein.”

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system, i.e., thedegree of precision required for a particular purpose, such as apharmaceutical formulation. For example, “about” can mean within 1 ormore than 1 standard deviations, per the practice in the art.Alternatively, “about” can mean a range of up to 20%, preferably up to10%, more preferably up to 5%, and more preferably still up to 1% of agiven value. Alternatively, particularly with respect to biologicalsystems or processes, the term can mean within an order of magnitude,preferably within 5-fold, and more preferably within 2-fold, of a value.Where particular values are described in the application and claims,unless otherwise stated, the term “about” meaning within an acceptableerror range for the particular value should be assumed.

Pathogenic Age-Associated B Cells, Their Regulation and Role inAutoimmune Disease

Age-associated B cells (ABCs) are a B cell subset, which exhibits uniquephenotypic and functional characteristics and can be regulated by T-bet.Although ABCs accumulate in autoimmune disorders, they can alsoaccumulate in non-autoimmune subjects. Additionally, some ABCs produceautoantibodies, and some do not. A detailed understanding of themolecular pathways that promote their expansion and function inautoimmune settings is largely unknown. However, as shown herein, someABCs are benign and some are pathogenic, causing autoimmune andlymphoproliferative diseases as well as chronic inflammatory disorders.The current invention is based upon the discovery of these pathogenicABCs (i.e., ABCs that cause or are associated with disease) and theproteins that regulate their expansion, function and differentiation.

In particular, pathogenic ABCs express CD11c (Example 2). Using doubleknockout mice for SWAP70 and DEF6 (SWEF deficient) which develop alupus-like syndrome, it was found that ABCs from these mice express manyof the cell markers found in ABCs from wild type mice including CD86,MHCII and IgM. However, these cells downregulated CD5, which othersreported that ABCs do express (see Rubtsova et al. 2015). Additionally,these pathogenic ABCs are detected at a much earlier age in the DKO micethan the benign ABCs found in wild type mice. These pathogenic ABCs alsosecrete anti-dsDNA IgG2c and other autoantibodies. See Example 2.

These pathogenic ABCs from the DKO mice exhibited an IL-21-dependentexpansion with pro-inflammatory capabilities, produced autoantibodies,and as compared to ABCs from wild type non-autoimmune female mice,displayed a distinctive transcriptome marked by increased Igtranscription and diminished expression of a subset of myeloid-relatedprograms. Chromatin accessibility profiles revealed a unique chromatinlandscape in ABCs from DKO mice, which is enriched in open chromatinregions containing IRF, AP1/BATF, and T-bet binding motifs but depletedin regions containing motifs targeted by PU.1 and MAF. Furthermore, itis shown herein that in the absence of SWEF proteins, IL-21 stimulationof B cells leads to dysregulated IRF5 activity and that the generationof pathogenic ABCs and lupus development in DKO female mice does notoccur. Thus, the generation of pathogenic ABCs is controlled at least inpart by IRF5 and SWEF proteins. Taken together these studies uncover anew genetic pathway that controls the generation of pathogenic ABCs inautoimmunity as well as the distinction between these pathogenic ABCsand those that accumulate normally, benign ABCs. See Examples 3-8.

The genome-wide transcriptional profiling demonstrated that the SWEFproteins regulate several key processes that can impact the expansionand function of ABCs. In particular, the GSEA pathway analysisidentified alterations in a number of pathways involved in the controlof cellular proliferation, which can play a crucial role in thepremature accumulation of ABCs in DKO mice. This analysis also revealeda marked ability of pathogenic ABCs to promote inflammation via theproduction of chemokines and cytokines. Thus, the contribution of ABCsto autoimmune pathophysiology is multifactorial and encompasses bothproduction of autoantibodies especially of the pathogenic IgG2a/cisotype and the ability to promote inflammation via recruitment ofinflammatory cells and production of pro-inflammatory cytokines. SeeExample 4.

The absence of SWEF proteins also resulted in increased binding of T-betto several ABC specific peaks suggesting cooperativity between IRF5 andT-bet at least for some ABC specific regulatory regions. The notion thatsuch cooperativity is at play was further reinforced by the decreasedbinding of T-bet to ABC-specific regions in the absence of IRF5 and bymutational analysis, which revealed an important role for the DNAbinding domain of IRF5 in the optimal recruitment of T-bet toABC-specific sites. See Example 7.

The enrichment in IRF motifs in DKO ABCs was mechanistically linked withan increase in the activity of IRF5 due to the lack of the inhibitoryeffects of the SWEF proteins. Coimmunoprecipitation experimentsdemonstrated that endogenous IRF5 can interact with both DEF6 andSWAP-70 supporting the idea that its activity can be inhibited by aheterodimer of the two SWEF proteins. Interaction of the SWEF proteinswith IRF5 was mediated by the IRF Association Domain (IAD) of IRF5 inline with the known ability of this family to interact with the IAD ofIRF4 and the presence of structural similarities between the IAD of IRF4and that of IRF5. Indeed, the SWEF proteins do not interact with IRF2,which carries a distinct type of IAD. See Example 7.

The work described herein now implicates IRF5 downstream of IL-21signaling thus positioning IRF5 as a common mediator of two keystimulatory pathways for ABC generation in autoimmune settings, IL21 andTLR7. The convergence of these two pathways onto IRF5 is likely tocontribute to the dramatic effect observed upon monoallelic deletion ofIRF5 on the development of disease in the model used herein (Example 8).Such strong gene dosage effects may be particular relevant for human SLEwhere IRF5 risk variants have been shown to result in alterations inIRF5 levels (Cham et al. 2012; Lazzari and Jefferies 2014).

In addition to enrichment for IRF and AP-1/BATF motifs, which could beobserved irrespective of whether DKO ABCs were compared to DKO FoBs orto WT ABCs, comparison of the chromatin landscapes of wild type and DKOABCs revealed that pathogenic ABCs also exhibited a marked loss ofaccessible chromatin regions containing PU.1, MAF, and C/EBP motifs.These epigenetic changes were associated with downregulation of MAF andMAFB expression but maintenance of PU.1 levels. Importantly, given theknown repressive role of PU.1 on the quantity of antibody production andplasma cell differentiation, selective depletion of PU.1-bound peakscould also lessen the PU.1-mediated inhibitory effects and directlycontribute to the increased levels of Ig transcription of DKO ABCs aswell as enhance their ability to differentiate into plasma cells uponexposure to additional environmental stimuli. Thus, the presence ofdysregulated IRF5 activity combined with the loss of PU.1-containingrepressive complexes could represent a critical mechanism employed byautoimmune-prone ABCs to bypass critical checkpoints governing thetransition of B cells into antibody secreting cells.

While most DKO ABCs express surface IgM, the ability of these cells toproduce anti-dsDNA IgG2c upon stimulation suggests that they can undergoclass switching and differentiate into PCs. Studies using Blimp1reporter DKO mice have indeed demonstrated the presence ofCD11c+CD19loBlimp1hi cells in the spleens of DKO female mice (Example9). These cells also express high levels of CD138 and IRF4 suggestingthat they represent a population of ABCs that has differentiated towardPCs.

One of the most striking features of the autoimmune syndrome thatdevelops in DKO mice is the finding that, as observed for human SLE,this disorder preferentially affects the female gender. InterestinglyABCs accumulate in both DKO female and male mice. Unlike pathogenic ABCsfrom DKO male mice, however, ABCs from DKO female mice readily secretedanti-dsDNA IgG2c antibodies upon TLR7 stimulation suggesting that thepathogenic potential of DKO ABCs differs in female and male mice.Remarkably, crossing DKO male mice to Yaa mice (which carry aduplication of TLR7 on the Y chromosome) leads to their ability toproduce anti-dsDNA IgG2c upon stimulation (Example 10).

Taken together, these studies have led to the hypothesis that ABCs canundergo further differentiation into CD11c+ PCs and that thedifferentiation of ABCs into CD11c+ PCs is regulated by sex-specificmechanisms. By investigating the transcriptional and epigenetic profilesof CD11c+ PCs from DKO mice as compared to CD11c− PCs, and examiningwhether IRF4 cooperates with IRF5 in regulating thedifferentiation/function of CD11c+ PCs, it will be shown that the CD11c+PC cells from DKO mice have a unique transcriptional and epigeneticprofile and that IRF4 and IRF5 regulate the differentiation of thepathogenic ABCs to PCs (Example 11). The contribution of sex-specificpathways to the differentiation/function of CD11c+ PCs will also beinvestigated (Example 12). Since aberrant B cell and PC homeostasis isone of the hallmarks of SLE and several lymphoproliferative diseases,these studies will provide fundamental insights into the molecularfeatures that characterize the PCs that expand in autoimmune andlymphoproliferative settings.

Thus the work reported herein shows that pathogenic ABC cells play arole in autoimmunity. Importantly these studies demonstrate that thereis a subset of CD11c+ cells that uniquely relies on the cooperation ofTbet and IRF5 for their expansion and function. The genome-wide analysisset forth herein demonstrates that these cells exhibit a uniquetranscriptional profile that is associated with a distinctive chromatinlandscape, especially when compared to non-pathogenic ABCs.Additionally, DEF6 and SWAP-70 regulate IRF5 activity thus controllingits accessibility to key target genes and its cooperativity with Tbet.

Furthermore, aberrancies and/or polymorphisms in IL-21, its receptor, orIRF5 have also been associated with several autoimmune disordersincluding rheumatoid arthritis and inflammatory bowel disease (Sarra etal. 2013; Eames et al. 2016). The dysregulation in the ability of theSWEF proteins to restrain IRF5 activity in response to IL-21 and,therefore, properly control ABC expansion and function, could alsocontribute to other autoimmune diseases.

Given that the ABCs are known to accumulate in non-autoimmune mice thesestudies also provide key information into the factors that regulate theexpansion of these cells into pathogenic ABCs. The identification ofthese factors controlling the expansion and function of pathogenic ABCcells, which include DEF6, SWAP-70, IRF5, IL-21, and a number of genes,also provides a method to develop new therapeutic targets fortherapeutic intervention for autoimmune, lymphoproliferative andaging-related diseases.

Inhibition of Interferon Regulatory Factor 5 (IRF5)

It has been discovered that the transcription factor, interferonregulatory factor 5, is necessary for ABCs to become pathogenic andcause autoimmune and lymphoproliferative disease. Thus, one embodimentof the current invention is a method of abolishing or reducingpathogenic ABCs in a subject in need thereof by administering atherapeutically effective amount of an agent that antagonizes, inhibitsor reduces the expression and/or activity of IRF5. A further embodimentof the current invention is a method of preventing and/or treating anautoimmune or lymphoproliferative disease by abolishing or reducingpathogenic ABCs in a subject in need thereof by administering atherapeutically effective amount of an agent that antagonizes, inhibitsor reduces the expression and/or activity of IRF5. Methods for reducingexpression of a protein are also well known in the art. Reduction ofIRF5 expression may be at the transcriptional, translational orpost-translational level.

IRF5 as used herein includes human IRF5, which is encoded by the humanIRF5 gene located at chromosome 7q32 (OMIM ID 607218). IRF5 is a memberof the IRF family; it is a transcription factor that possesses ahelix-turn-helix DNA-binding motif and mediates virus- and interferon(IFN)-induced signaling pathways. It is appreciated that severalisoforms/transcriptional variants of IRF5 are known. Preferably, theinhibitor of IRF5 inhibits at least the expression or activity of anyhuman IRF5 variant. It is also well known that IRF5 is polymorphic, anda large number of polymorphisms, including SNPs are known. Thus, in anembodiment, the inhibitor of IRF5 also inhibits expression or activityof naturally-occurring variants of human IRF5 in which one or more ofthe amino acid residues have been replaced with another amino acid.

One agent for inhibition of IRF5 is a small molecule.

Additional inhibitors of IRF5 expression and activity includeIRF5-specific RNAi, IRF5-specific short RNA, IRF5-specific antisense(e.g., IRF5-specific morpholinos) and triplet-forming oligonucleotides,and IRF5-specific ribozymes.

Short RNA molecules include short interfering RNA (siRNA), smalltemporal RNAs (stRNAs), short hairpin RNA (shRNA), and micro-RNAs(miRNAs). Short interfering RNAs silence genes through an mRNAdegradation pathway, while stRNAs and miRNAs are approximately 21 or 22nt RNAs that are processed from endogenously encoded hairpin-structuredprecursors, and function to silence genes via translational repression.See, e.g., McManus et al. (2002). RNA 8(6):842-50; Morris et al. (2004).Science 305(5688):1289-92; He and Hannon. (2004). Nat. Rev. Genet.5(7):522-31. IRF5 siRNA are commercially available, for example, asOn-target SMMRT pool reagents from Dharmacon, USA (catalogue No.L-011706-00-0005), and from Santa Cruz Biotechnology, USA (catalogue No.sc-72044).

“RNA interference, or RNAi” a form of post-transcriptional genesilencing (“PTGS”), describes effects that result from the introductionof double-stranded RNA into cells (reviewed in Fire. (1999). TrendsGenet. 15:358-363; Sharp. (1999) Genes Dev. 13:139-141; Hunter. (1999).Curr. Biol. 9:R440-R442; Baulcombe. (1999). Curr. Biol. 9:R599-R601;Vaucheret et al. (1998). Plant J. 16:651-659). The active agent in RNAiis a long double-stranded (antiparallel duplex) RNA, with one of thestrands corresponding or complementary to the RNA which is to beinhibited. The inhibited RNA is the target RNA. The long double strandedRNA is chopped into smaller duplexes of approximately 20 to 25nucleotide pairs, after which the mechanism by which the smaller RNAsinhibit expression of the target is largely unknown at this time. WhileRNAi was shown initially to work well in lower eukaryotes, for mammaliancells, it was thought that RNAi might be suitable only for studies onthe oocyte and the preimplantation embryo.

More recently, it was shown that RNAi would work in human cells if theRNA strands were provided as pre-sized duplexes of about 19 nucleotidepairs, and RNAi worked particularly well with small unpaired 3′extensions on the end of each strand (Elbashir et al. (2001). Nature411:494-498). In this report, “short interfering RNA” (siRNA, alsoreferred to as small interfering RNA) were applied to cultured cells bytransfection in oligofectamine micelles. These RNA duplexes were tooshort to elicit sequence-nonspecific responses like apoptosis, yet theyefficiently initiated RNAi. Many laboratories then tested the use ofsiRNA to knock out target genes in mammalian cells. The resultsdemonstrated that siRNA works quite well in most instances.

For purposes of reducing the activity of IRF5, siRNAs to the geneencoding IRF5 can be specifically designed using computer programs.Illustrative nucleotide sequences encoding the amino acid sequences ofthese components are readily available.

Software programs for predicting siRNA sequences to inhibit theexpression of a target protein are commercially available and find use.One program, siDESIGN from Dharmacon, Inc. (Lafayette, Colo.), permitspredicting siRNAs for any nucleic acid sequence, and is available on theinternet at dharmacon.com. Programs for designing siRNAs are alsoavailable from others, including Genscript (available on the internet atgenscript.com/ssl-bin/app/rnai) and, to academic and non-profitresearchers, from the Whitehead Institute for Biomedical Research foundon the worldwide web at“jura.wi.mit.edu/pubint/http://iona.wi.mit.edu/siRNAext/.”

Alternatively, double-stranded (ds) RNA is a powerful way of interferingwith gene expression in a range of organisms that has recently beenshown to be successful in mammals (Wianny and Zernicka-Goetz. (2002),Nat. Cell. Biol. 2:70-75). Double stranded RNA corresponding to thesequences of a IRF5 polynucleotides can be introduced into or expressedin cells of a candidate organism to interfere with IRF5 activity.

MicroRNA can also be used to inhibit IRF5. MicroRNAs are smallnon-coding RNAs averaging 22 nucleotides that regulate the expression oftheir target mRNA transcripts by binding. Binding of microRNAs to theirtargets is specified by complementary base pairing between positions 2-8of the microRNA and the target 3′ untranslated region (3′ UTR), an mRNAcomponent that influences translation, stability and localization.Additionally, this microRNA can also be modified for increasing otherdesirable properties, such as increased stability, decreased degradationin the body, and increased cellular uptake.

Ribozymes are RNA molecules capable of cleaving targeted RNA or DNA.Examples of ribozymes are described in, for example, U.S. Pat. Nos.5,180,818; 5,168,053; 5,149,796; 5,116,742; 5,093,246; and 4,987,071,all incorporated herein by reference. Ribozymes specific for IRF5 can bedesigned by reference to the IRF5 cDNA sequence.

A further approach is to express anti-sense constructs directed againstthe polynucleotides of IRF5 to inhibit gene function and to abolish ordecrease pathogenic ABCs.

Antisense oligonucleotides are single-stranded nucleic acids, which canspecifically bind to a complementary nucleic acid sequence. By bindingto the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNAduplex is formed. By binding to the target nucleic acid, antisenseoligonucleotides can inhibit the function of the target nucleic acid.Typically, antisense oligonucleotides are 15 to 35 bases in length.However, it is appreciated that it may be desirable to useoligonucleotides with lengths outside this range, for example 10, 11,12, 13, or 14 bases, or 36, 37, 38, 39 or 40 bases. Thus, with knowledgeof the IRF5 cDNA sequence, polynucleotide inhibitors of IRF5 expressioncan be produced using methods well known in the art.

The antisense molecules may be expressed from any suitable geneticconstruct and delivered to the subject. Typically, the genetic constructwhich expresses the antisense molecule comprises at least a portion ofthe IRF5 cDNA or gene operatively linked to a promoter which can expressthe antisense molecule in the cell. Preferably, the genetic construct isadapted for delivery to a human cell.

Other agents would include antibodies to the components of IRF5. Suchantibodies are commercially available or can be produced by methodsknown in the art.

The terms “antibody” and “antibodies” include polyclonal antibodies,monoclonal antibodies, humanized or chimeric antibodies, single chain Fvantibody fragments, Fab fragments, and F(ab′)₂ fragments. Polyclonalantibodies are heterogeneous populations of antibody molecules that arespecific for a particular antigen, while monoclonal antibodies arehomogeneous populations of antibodies to a particular epitope containedwithin an antigen. Monoclonal antibodies and humanized antibodies areparticularly useful in the present invention.

Antibody fragments that have specific binding affinity for a target ofinterest can be generated by known techniques. Such antibody fragmentsinclude, but are not limited to, F(ab′)₂ fragments that can be producedby pepsin digestion of an antibody molecule, and Fab fragments that canbe generated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed. Single chainFv antibody fragments are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge (e.g., 15 to 18amino acids), resulting in a single chain polypeptide. Single chain Fvantibody fragments recognizing a target of interest can be producedthrough standard techniques, such as those disclosed in U.S. Pat. No.4,946,778.

It is also well known that IRF5 is polymorphic, and a large number ofpolymorphisms, including SNPs are known. Thus, in an embodiment, theinhibitor of IRF5 also inhibits at least one function or activity ofnaturally-occurring variants of human IRF5 in which one or more of theamino acid residues have been replaced with another amino acid.

It is also appreciated that the IRF5 inhibitor may be one that inhibitsat least one function or activity of an orthologue of IRF5 in anotherspecies, for example IRF5 from a horse, dog, pig, cow, sheep, rat,mouse, guinea pig or a primate. It will be appreciated, that when theinhibitor is administered to a particular individual, the inhibitor isone that modulates at least one function or activity of IRF5 from thesame species as that individual. Thus, when the patient is a humanpatient, the inhibitor inhibits at least one function or activity ofhuman IRF5, and so on.

Methods and routes of administering polynucleotide inhibitors, such assiRNA molecules, antisense molecules and ribozymes, to a patient, arewell known in the art and described in more detail below. It isappreciated that polynucleotide inhibitors of IRF5 may be administereddirectly, or may be administered in the form of a polynucleotide thatencodes the inhibitor. Thus, as used herein, unless the context demandsotherwise, by administering to the individual an inhibitor of IRF5 whichis a polynucleotide, includes the meanings of administering theinhibitor directly, or administering a polynucleotide that encodes theinhibitor, typically in the form of a vector.

In a further embodiment, the inhibitor may be a dominant-negative mutantof IRF5. As well as those mentioned above, the dominant-negative mutantmay have a mutated or deleted DNA binding domain (DBD). Specificexamples of mutations that have dominant-negative effect include amutation at Alanine at position 68, especially when substituted withProline, which results in complete loss of DNA binding activity (Yang etal. (2009). Plos One v4(5):e5500). Suitable methods, routes andcompositions for preparing polypeptide inhibitors of IRF5 and nucleicacid molecules that encode them and administering them to a patient areknown in the art and described below, and include viral vectors such asadenoviral vectors.

Agonizing, Activating or Increasing SWEF Proteins

As discussed above, the current invention is based upon the discoverythat reducing the aberrant expansion of pathogenic ABCs depend on twoSWEF proteins, SWAP70 and DEF6. Thus, increasing the expression and/oractivity of these proteins can reduce or abolish pathogenic ABCs.Methods for increasing expression and/or activity of a protein are alsowell known in the art. Increasing SWEF expression may be at thetranscriptional, translational or post-translational level.

Thus, a further embodiment of the current invention is a method ofabolishing or reducing pathogenic ABCs in a subject in need thereof byadministering a therapeutically effective amount of an agent thatagonizes, activates or increases the expression and/or activity ofSWAP-70 and/or DEF6. Such agents that can be used in this method includebut are not limited to agents for increasing the expression of the geneencoding SWAP-70 and/or DEF6 and include nucleic acids which encode theSWAP-70 and/or DEF6 proteins, or the entire SWAP-70 and/or DEF6 gene, ora nucleic acid that is substantially homologous to the SWAP-70 and/orDEF6 genes, or a variant, mutant, fragment, homologue or derivative ofthe SWAP-70 and/or DEF6 genes that produces a protein that maintains orincreases their function.

The gene or a nucleic acid which encodes the SWAP-70 and/or DEF6proteins, or a nucleic acid that is substantially homologous to theSWAP-70 and/or DEF6 genes, or a variant, mutant, fragment, homologue orderivative of the SWAP-70 and/or DEF6 genes that produce proteins withmaintained or increased function can also be used in the methods of theinvention.

The sequences of human SWAP-70 and DEF6 are available on the NationalCenter for Biotechnology Database and can be used to manufacturevariants, mutants, fragments, homologues and derivatives which maintainor have increased function.

DNA or other nucleic acids such as mRNA can also be used in the method.

While it would be understood that any agent or agents that increase orupregulate the expression of SWAP-70 and/or DEF6, would also most likelyincrease SWAP-70 and/or DEF6 proteins, alternatively, an agent or agentsthat directly increase or promote the activation, amount and/or activityof the proteins can be used in the methods.

Alternatively, administering the proteins can be used in the methods.This includes the administration of a polypeptide, or a variant thereofhaving at least 90% sequence identity with the SWAP 70 and/or DEF6polypeptides.

In an embodiment, the variant of the polypeptide has at least 91%sequence identity, or at least 92% sequence identity, or at least 93%sequence identity, or at least 94% sequence identity, or at least 95%sequence identity, or at least 96% sequence identity, or at least 97%sequence identity, or at least 98% sequence identity, or at least 99%sequence identity, with the sequence of the polypeptide of which it is avariant. Thus, preferably, the variant of the polypeptide has at least91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with thesequence of the SWAP70 and/or DEF6 polypeptide. Such variants may bemade, for example, using the methods of recombinant DNA technology,protein engineering and site-directed mutagenesis, which are well knownin the art, and discussed in more detail below.

The percent sequence identity between two polypeptides may be determinedusing suitable computer programs.

Polypeptides, may be prepared using an in vivo or in vitro expressionsystem. Preferably, an expression system is used that provides thepolypeptides in a form that is suitable for pharmaceutical use, and suchexpression systems are known to the skilled person. As is clear to theskilled person, polypeptides of the invention suitable forpharmaceutical use can be prepared using techniques for peptidesynthesis.

A nucleic acid molecule encoding, for example, the proteins or variantsthereof, may be used to transform a host cell or host organism forexpression of the desired polypeptide. Suitable hosts and host cells areknown in the art and may be any suitable fungal, prokaryotic oreukaryotic cell or cell line or organism, for example: bacterialstrains, including gram-negative strains such as Escherichia coli andgram-positive strains such as Bacillus subtilis or of Bacillus brevis;yeast cells, including Saccharomyces cerevisiae; or Schizosaccharomycespombe; amphibian cells such as Xenopus oocytes; insect-derived cells,such SF9, Sf21, Schneider and Kc cells; plant cells, for example tobaccoplants; or mammalian cells or cell lines, CHO-cells, BHK-cells (forexample BHK-21 cells) and human cells or cell lines such as HeLa, aswell as all other hosts or host cells that are known and can be used forthe expression and production of polypeptides.

The polypeptides or variants thereof, may be made by chemical synthesis,again using methods well known in the art for many years. In certainembodiments, polypeptides for administration to a patient may be in theform of a fusion molecule in which the polypeptide is attached to afusion partner to form a fusion protein. Many different types of fusionpartners are known in the art. One skilled in the art can select asuitable fusion partner according to the intended use of the fusionprotein. Examples of fusion partners include polymers, polypeptides,lipophilic moieties, and succinyl groups. Certain useful protein fusionpartners include serum albumin and an antibody Fc domain, and certainuseful polymer fusion partners include, but are not limited to,polyethylene glycol, including polyethylene glycols having branchedand/or linear chains. In certain embodiments, the polypeptide may bePEGylated, or may comprise a fusion protein with an Fc fragment.

In an embodiment, the polypeptide may be fused to or may compriseadditional amino acids in a sequence that facilitates entry into cells(i.e. a cell-penetrating peptide). Thus, for example, the SWAP70, DEF6or variant thereof or a polypeptide may further comprise the sequence ofa cell-penetrating peptide (also known as a protein transduction domain)that facilitates entry into cells. As is well known in the art,cell-penetrating peptides are generally short peptides of up to 30residues having a net positive charge and act in a receptor-independentand energy-independent manner.

Additionally or alternatively, the polypeptide may be fused to or maycomprise additional amino acids in a sequence that facilitates entryinto the nucleus (i.e., a nuclear localization sequence (NLS), akanuclear localization domain (NLD)). Thus, for example, the SWAP70 orDEF6 protein or variant thereof may further comprise the sequence of anNLS that facilitates entry into the nucleus. NLS includes anypolypeptide sequence that, when fused to a target polypeptide, iscapable of targeting it to the nucleus. Typically, the NLS is one thatis not under any external regulation (e.g. calcineurin regulation) butwhich permanently translocates a target polypeptide to the nucleus.

It is appreciated that the sequence of the cell-penetrating peptideand/or the NLS may be adjacent to the sequence of the protein orvariant, or these sequences may be separated by one or more amino acidsresidues, such as glycine residues, acting as a spacer.

Therapeutic proteins produced as an Fc-chimera are known in the art. Forexample, Etanercept, the extracellular domain of TNFR2 combined with anFc fragment, is a therapeutic polypeptide used to treat autoimmunediseases, such as rheumatoid arthritis.

In certain embodiments, the fusion partner may be a polymer, forexample, polyethylene glycol (PEG). PEG may comprise branched and/orlinear chains. In certain embodiments, a fusion partner comprises achemically-derivatised polypeptide having at least one PEG moietyattached.

The fusion partner may be attached, either covalently or non-covalently,to the amino-terminus or the carboxy-terminus of the polypeptide. Theattachment may also occur at a location within the polypeptide otherthan the amino-terminus or the carboxy-terminus, for example, through anamino acid side chain (such as, for example, the side chain of cysteine,lysine, histidine, serine, or threonine).

Modulating Genes that Upregulated and Downregulated in Pathogenic ABCs

As shown herein, certain genes are upregulated and/or enriched inpathogenic ABCs and some are downregulated.

Thus, one embodiment of the current invention is a method of abolishingor reducing pathogenic ABCs in a subject in need thereof byadministering a therapeutically effective amount of an agent thatantagonizes, inhibits or reduces the expression and/or activity ofcertain upregulated genes. A further embodiment of the current inventionis a method of preventing and/or treating an autoimmune orlymphoproliferative disease by abolishing or reducing pathogenic ABCs ina subject in need thereof by administering an therapeutically effectiveamount of an agent that antagonizes, inhibits or reduces the expressionand/or activity of certain upregulated genes.

In particular, 111 genes listed in Table 1 were upregulated inpathogenic or DKO ABCs as compared to WT ABCs. One or more of theseupregulated genes would be a target for reduction of expression in orderto reduce or abolish pathogenic ABCs.

More specifically, the methods of the invention include the inhibitionof one or more genes including but not limited to: Cxcl9, Cxcl10, Ccl4,Ccl5, Ccl8, Il1r2, Li2rb2, Il18r1, Il18rap, Csf1, Tbx21, Itgax, Itgam,Ctla4, Sema3d, Sema4c, Bmp6, Itga8, Cc122, Tnsfsf4, Cxcr3, Ccr1, Plxnd1,Itgb1, Ifnγ, Il6, Runx, MyoG, NF-kB, stat5, Hbp1, Srebf1, Zbtb32, Nfil3,IL-12a, CD28, CD9, FcRL5, CD30/CD30L, c-kit, CD15, CD244, CD68, Lmo7,Tnip3, Msc, Mist1, Id2, Insm1, TNFa, Thsd7a, IL-13/IL-13Ra1, IL-4, IL-5,and NDNF.

Other genes whose expression and/or activity are upregulated inpathogenic ABCs as compared to benign ABCs include but are not limitedto Stat5, Hbp1, Srebf1, Zbtb32, LifR, AP1 family members and Batf familymembers. These genes would also be considered targets for reduction ofexpression in order to reduce or abolish pathogenic ABCs.

Most specifically, one or more genes related to higher immunoglobulinproduction including but not limited to Nfil3, Jun, and IL-9/IL-9R,would be targets for reduction of expression in order to reduce orabolish pathogenic ABCs.

The inhibition of these genes can be accomplished by any method known inthe art including but not limited to the ones described above fordecreasing expression of the gene that encodes IRF5, including but notlimited to small molecules, RNAi, short RNA, antisense (e.g.,morpholinos) and triplet-forming oligonucleotides, and ribozymes.

A further embodiment of the current invention is a method of abolishingor reducing pathogenic ABCs in a subject in need thereof byadministering a therapeutically effective amount of an agent thatagonizes, activates or increases the number, expression and/or activityof certain downregulated genes. A further embodiment of the currentinvention is a method of preventing and/or treating an autoimmune orlymphoproliferative disease by abolishing or reducing pathogenic ABCs ina subject in need thereof by administering a therapeutically effectiveamount of an agent that agonizes, activates or increases the number,expression and/or activity of certain downregulated genes. This could beaccomplished by introducing a nucleic acid encoding the gene or aportion into the subject as described above for increasing SWEFproteins.

In particular, the genes listed in Table 2 were downregulated inpathogenic or DKO ABCs as compared to WT ABCs. One or more of thesedownregulated genes would be a target for an increase of expression inorder to reduce or abolish pathogenic ABCs. More specifically, genesthat are downregulated in pathogenic ABCs include but are not limited toMafA, MafB, c-maf, Mertk, Cebp, Rora, Bcl6, Pxk, Smad1, Emp2, Pouf2f2,PU.1, Rel, Foxj3, Hand1, Cebp, Rora, Prdm1, Spic, and Pparg. Morespecifically, one or more genes related to myeloid-related genesincluding but not limited to AHR and PPARGc1a would be targets for anincrease of expression in order to reduce or abolish pathogenic ABCs.

TABLE 1 Genes Upregulated in Pathogenic (DKO) ABCs Genes Genes GenesGenes Gm9825 Nfil3 Socs1 Slc25a19 Gdpd3 Igkv8-21 Serpina3f 9330175E14RikHmga1-rs1 Igkv3-10 Ighv1-61 C920025E04Rik Gm4841 Igkv4-80 Gm16710 Glo1Thsd7a Gm10505 Tmem176b Gatsl3 H2-T10 Igkv17-121 Cplx2 Fgl2 Adm Ighv14-3Trp73 Gas7 Ighe Igkv4-68 Tmem176a Egr2 Ndnf Hspg2 Slc30a4 Il2ra Igkv5-45Il12a Ndrg1 Zbtb32 Lifr Ighv5-16 Jun Csf1 Ighv1-84 Igkv6-20 Camkk1Igkv16-104 Igkv3-2 Ighv3 -6 Iigp1 Fscn1 Ighg1 Sox5 Nostrin Tmem231 Dnah8Ighv9-2 Lrrk2 H2-T24 Ighv5-2 Il9r Il4i1 Gnb4 Igkv17-127 Gramd2 Eml5 LipcTagap1 Ffar2 H2-Q6 Havcr1 Ighv1-85 Slc22a15 Prr5l Akap5 Zfp365 Igkv6-25Csf2rb2 Mical3_1 Tnip3 Igkv13 -84 Nlrc3 Hipk2 Wdfy1 Plcg1 Plscr1 Lmo7Igkv6-14 Gadd45g Pdcd1lg2 Pmepa1 Pard3b Wee1 Ermard Aox4 Lamp3 OsmIgkv4-74 Nrp2 Ighv1-52 Igkv14-126 Gm2619 Rec8 Igkv3-5 Myo3b Igkv11-125Ighv9-1 Chst7 Trio

TABLE 2 Genes Downregulated in Pathogenic (DKO) ABCs Genes Genes GenesGenes Genes Genes Rnaset2b Dnase1l3 Slc37a2 Dennd2d Pld3 Calcrl Rap2aGclm Kcnk6 Nr4a1 Ighv5 -9 Itga2 Hpcal1 Tns3 Myo10 Slc35f6 Igkv6-13 Gpm6b4632428N05Rik P2rx4 Dram2 Cr2_1 Plekhg3 Smad1 Mcfd2 Cfp Tmem206 Esr1Rnf149 Rtp4 Rnps1 8430419L09Rik Nagk Igkv1-132 Gna12 Arl4d Spred1 Adrb2Lidlr Arhgap18 Rasgef1b Tmem51 Fam105a Il6ra Mid1 Erlin2 Megf8 S100a4Zfp36l2 Lpin1 Arhgap19 Stard8 Dip2c Hmga1 Asah2 Por Glul Megf9 Plcl1Il13ra1 Akr1b10 Wwp1 Lag3 Abcc5 Blvra Hsd11b1 Il6st Dmxl2 Slc29a1Rab11fip5 Swap70 Itsn1 Cebpb Galnt7 Arrb2 Marveld1 Fam149a Cmtm3 Arrdc3Ubtd1 Ppap2a Pik3cb Ifnlr1 Osbpl1a Cyb5a Cttn Asah1 Pcyox1 Bmf Lpar5P4ha1 Etv5 Hexa Nfix Lmbrd2 Efnb1 Leprot Hip1 Comt Ece1 Alpl Pira2Slc7a7 Ets2 Tlr4 Paqr4 Lcp2 Pvrl4 Apobec2 Gm4951 Ifih1 Fam26f Clec7a_1Cdld1 Mpp6 Eno3 Cd63 RP23-458B6.6 Mcoln2 Skp2 Hes1 Gna15 Emr1 Slc46a11190002N15Rik Abcb4 Slc43a2 St3gal4 Nucb2 Fhod1 Mllt4 Gstm1 Hs6st1Csf2ra Rab6b Rbpms Slc48a1 Mpeg1 Ccdc88b Lgals3bp Slc8b1 Tlr3 Tcn2 SmagpLgmn Sh2d1b2 Fez2 Spon1 Slc15a2 Ftl1 Plin2 Man1c1 Dfna5 Gab2 Kcnk13Slc1a3 Tenm4 Nxpe4 Plxna1 Tmem65 Gm13994 Mgst1 Hist1h1c Kif13a Pla2g7Nxpe5 Ppm1h Sgk1 Rab20 C5ar2 Rnf150 Rgl1 Ms4a7 C3 Basp1 Lipa A4galtAngptl4 Abcg3 B430306N03Rik Prkra Dnajb13 1700025G04Rik E330020D12RikCrisp3 Lst1 Pkp4 Sema4c Rab32 Nfam1 Fcgrt Ckb Itfg3 Ppap2b Gsr AoahAcer3 Ctnnd1 Rasa4 Syt15 Aph1b Marcks Tyrobp Slamf8 Gsto1 Vamp5 AsphNinj1 Cadm1 Tbc1d4 Map4k3 Ms4a6d P2rx7 Parp12 Sirpa Mcoln3 Arhgap39Hist1h2bc Akr1c13 Pla2g15 Slc9a9 Epb4.1l1 Igsf8 Acot11 Lima1 Galc Btnl6Pla2g4a Sh3bgrl2 Fads1 Myo9a Fcgr4 Smox Gas6 Id2 Rnase4 Klk1 Scamp1 Ccl5Dram1 Slc12a2 4931406C07Rik Cyp27a1 Hpgds Lyz2 Ifitm2 Slc16a7 Myof Idh1Tmem141 Osgin1 Ggt5 Pde2a Fam102b Cln8 Crim1 Lpcat2 Camk1 Plxnb2 Gbp8Mitf Mical2 Ppt2 Avpi1 Lrp8 Ighv1-7 Tmem26 Tppp Ccnd1 Cd200r4 Tle1 Car2Ctsl Abcb1a Sdc3 Scn1b Dhrs3 Rsad2 Slc39a8 Fam213b Ctsb Cd5l Cd36 DseScarb1 Anpep Serpinb6a Bmpr2 Cyb5r1 Hsd3b7 Fabp4 Pstpip2 Il11ra1 Asb2Cpq Mt1 Fblim1 Ctsd Igha Lrrc25 Kdelc2 Tbc1d8 Cmtm4 Metrnl Cxcl9 CtsfMgat4b Plod1 Dab2ip Cd200r1 Pald1 Frmd4a Rragd Slc12a7 Hnrnpll PtafrLrp12 Ptgs1 1830077J02Rik Cd68 Abca9 Slc8a1 Cd244 Adam23 Timp2 Anxa3 NplDlg2 Hk3 Sirpb1b Arsg Def6 Fcer1g Itga9 1l18 Tbxas1 Tspan15 Tifab Actn1Tlr13 Snx24 B3galnt1 Creg1 Tmem86a Gpr35 Fcgr1 Dock4 Havcr2 Parvb Sepp1Tlr8 Cdc42ep2 Arhgap32 Sash1 Rgs10 Tnfrsf1a Fcgr3 Fpr1 Pla2g2d C1qaIl18bp Trim47 Sall2 Pdgfc Cd302 Ltbr Sema6d Stk39 Lair1 C6 Lrp5 C1qcSiglech Ppfia4 Clec4a3 Tgfbi AF251705 Trf Tnfrsf21 Ubd Lilra5 Dmpk Fgd4Dock5 Cnksr3 Tbc1d2b Slc4a1 Clec4a1 Nr1h3 Pdgfb Soga1 Sirpb1a Acot1Itgad Agap1 Epb4.1l3 Gdpd1 Dock7 Amz1 Kcnj2 Aatk Kcna2 Ifitm3 RuaClec12a Itgb5 Hba-a1 Rab3il1 Ccl6 Fmnl2 Raver2 Mafb Acp2 Alas2 AI6078736430548M08Rik Adamdec1 Jup Gtf2ird1 Pygl Cebpa Cttnbp2nl Iqgap2 Aif1Pak1 Pparg Nuak1 Pigz F11r Adrbk2 Wdfy3 Mrc1 Matn2 Ttyh2 Lrp1 Abcd2Siglece Cmklr1 Ifi204 Nptxr Fzd7 Igsf6 Clec1b Hba-a2 Adap2 Dgat2 Csf3rSqrdl Pid1 Pdlim4 Tgm2 Spic Slc16a10 Snta1 Scamp5 Fyb Large Rin2 Unc5aRims3 Oaf Klrk1 Adam22 Tns1 Hap1 Ear2 Tjp1 Maf Enpp4 Klra2 Slc22a23 TfecC1qb Tsku Tcf7l2 Serpinb9b Vnn3 Adrb1 Ptgis Slc7a8 Ptprm Pilrb2 Clec4nCcl24 Cd4 Cela1 Cd300e 1810011H11Rik Nfasc Hfe Slc11a1 Hmox1 ApobrA530099J19Rik Dgki Kcnj10 Rnf144b Cmbl Trpm2 Dysf Axl Mrap Aph1c Nos1Igf1 Hs3st2 Hbb-bs Apoc1 Slco2b1 Nav1_1 Ccdc148 Gm5150 Treml4 Gpd1 Nlrp3Frmd4b Fkbp9 Slc40a1 Clec4a2 Gfra2 Slc16a9 Gsta4 Tgm1 Ccr3 Pilrb1 Enpp2Hebp1 Kcnj16 Vcam1 Slc45a3 Hbb-bt Hpgd Gzma B4galt4 Tspan4 Gm13710 PilraAkr1b7 Cystm1 Lrp4 St6galnac2 Agmo Kctd12b Ptplad2 Abcc3 Tspan9 Sulf2Cd14 Galnt3 RP24-247B20.1 Paqr9 Mpzl1 Gm4980 Kitl Mertk Sort1 Cd300ldApol7c Prkar1b Slc7a2 Hcar2 Lphn3 Nid2 Csf1r Fcna Sdc2 Tnfaip2 Stab2Emr4 Glis3 Mras Vwf Epor Gpr141 Erbb2 Clec4b1 Pcolce2 Cd163 Tmem37Cd300a Vstm4 Rps13 Tnfrsf11a Crip2 Dlc1 Col14a1Administration of Agents

When the SWAP-70, DEF6, or IRF5 inhibitor, or inhibitor or activator ofa misregulated gene, is a nucleic acid such as DNA, RNA, interfering RNAor microRNA, methods for delivery include receptor mediated endocytosiswhere the nucleic acid is coupled to a targeting molecule that can bindto a specific cell surface receptor, inducing endocytosis and transferof the nucleic acid into cells. Coupling is normally achieved bycovalently linking poly-lysine to the receptor molecule and thenarranging for (reversible) binding of the negatively charged DNA or RNAto the positively charged poly-lysine component. Another approachutilizes the transferrin receptor or folate receptor which is expressedin many cell types. When producing the microRNA for this method ofadministration, the microRNA could be manufactured to have a guidestrand which is identical to the microRNA of interest and a passengerstrand that is modified and linked to a molecule for increasing cellularuptake

Another method to administer the nucleic acid to the proper tissue isdirect injection/particle bombardment, where the nucleic acid is beinjected directly with a syringe and needle into a specific tissue, suchas muscle.

An alternative direct injection approach uses particle bombardment(‘gene gun’) techniques: nucleic acid is coated on to metal pellets andfired from a special gun into cells. Successful gene transfer into anumber of different tissues has been obtained using this approach. Suchdirect injection techniques are simple and comparatively safe.

Another method for delivery of nucleic acid to the proper tissue or cellis by using adeno-associated viruses (AAV). Nucleic acid is delivered inthese viral vectors is continually expressed, replacing the expressionof the DNA or RNA that is not expressed in the subject. Also, AAV havedifferent serotypes allowing for tissue-specific delivery due to thenatural tropism toward different organs of each individual AAV serotypeas well as the different cellular receptors with which each AAV serotypeinteracts. The use of tissue-specific promoters for expression allowsfor further specificity in addition to the AAV serotype.

Other mammalian virus vectors that can be used to deliver the DNA or RNAinclude oncoretroviral vectors, adenovirus vectors, Herpes simplex virusvectors, and lentiviruses.

Liposomes are spherical vesicles composed of synthetic lipid bilayerswhich mimic the structure of biological membranes. The nucleic acid tobe transferred is packaged in vitro with the liposomes and used directlyfor transferring the nucleic acid to a suitable target tissue in vivo.The lipid coating allows the nucleic acid to survive in vivo, bind tocells and be endocytosed into the cells. Cationic liposomes (where thepositive charge on liposomes stabilize binding of negatively chargedDNA), have are one type of liposome.

The nucleic acid can also be administered with a lipid to increasecellular uptake. The nucleic acid may be administered in combinationwith a cationic lipid, including but not limited to, lipofectin, DOTMA,DOPE, and DOTAP.

Other lipid or liposomal formulations including nanoparticles andmethods of administration have been described as for example in U.S.Patent Publication 20030203865, 2002/0150626, 2003/0032615, and2004/0048787. Methods used for forming particles are also disclosed inU.S. Pat. Nos. 5,844,107, 5,877,302, 6,008,336, 6,077,835, 5,972,901,6,200,801, and 5,972,900.

The polypeptide or nucleic acid molecule for administration to thepatient may be formulated as a nanoparticle. Nanoparticles are acolloidal carrier system that has been shown to improve the efficacy ofan encapsulated drug by prolonging the serum half-life.Polyalkylcyanoacrylates (PACAs) nanoparticles are a polymer colloidaldrug delivery system that is in clinical development (described, forexample, by Stella et al. (2000) J. Pharm. Sci., 89: 1452-1464; Briggeret al. (2001) Int. J. Pharm 214: 37-42; Calvo et al. (2001) Pharm. Res.18: 1157-1166; and Li et al. (2001) Biol. Pharm. Bull. 24: 662-665).Biodegradable poly(hydroxyl acids), such as the copolymers ofpoly(lactic acid) (PLA) and poly(lactic-co-glycolide) (PLGA) are beingextensively used in biomedical applications and have received FDAapproval for certain clinical applications. In addition, PEG-PLGAnanoparticles have many desirable carrier features including (i) thatthe agent to be encapsulated comprises a reasonably high weight fraction(loading) of the total carrier system; (ii) that the amount of agentused in the first step of the encapsulation process is incorporated intothe final carrier (entrapment efficiency) at a reasonably high level;(iii) that the carrier has the ability to be freeze-dried andreconstituted in solution without aggregation; (iv) that the carrier bebiodegradable; (v) that the carrier system be of small size; and (vi)that the carrier enhances the particles persistence. Nanoparticles maybe synthesized using virtually any biodegradable shell known in the art.In one embodiment, a polymer, such as poly(lactic-acid) (PLA) orpoly(lactic-co-glycolic acid) (PLGA) is used. Such polymers arebiocompatible and biodegradable, and are subject to modifications thatdesirably increase the photochemical efficacy and circulation lifetimeof the nanoparticle. In one embodiment, the polymer is modified with aterminal carboxylic acid group (COOH) that increases the negative chargeof the particle and thus limits the interaction with negatively chargednucleic acids. Nanoparticles may also be modified with polyethyleneglycol (PEG), which also increases the half-life and stability of theparticles in circulation. Alternatively, the COOH group may be convertedto an N-hydroxysuccinimide (NHS) ester for covalent conjugation toamine-modified compounds.

Other protein modifications to stabilize a polypeptide, for example toprevent degradation, as are well known in the art may also be employed.Specific amino acids may be modified to reduce cleavage of thepolypeptide in vivo. Typically, N- or C-terminal regions are modified toreduce protease activity on the polypeptide. A stabilizing modificationis any modification capable of stabilizing a protein, enhancing the invitro half life of a protein, enhancing circulatory half life of aprotein and/or reducing proteolytic degradation of a protein. Forexample, polypeptides may be linked to the serum albumin or a derivativeof albumin. Methods for linking polypeptides to albumin or albuminderivatives are well known in the art.

It is appreciated that the compounds for administration to a patient,for example as described above, will normally be formulated as apharmaceutical composition, i.e. together with a pharmaceuticallyacceptable carrier, diluent or excipient.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human, and approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. “Carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas saline solutions in water and oils, including those of petroleum,animal, vegetable, or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil, and the like. A saline solution is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol, and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents.

Preferred methods of administration include oral; mucosal, such asnasal, sublingual, vaginal, buccal, or rectal; parenteral, such assubcutaneous, intravenous, bolus injection, intramuscular, orintraarterial; or transdermal administration to a subject. Mostpreferred method of administration are parenteral and oral.

These administrations can be performed using methods standard in theart. Oral delivery can be performed by complexing a therapeuticcomposition of the present invention to a carrier capable ofwithstanding degradation by digestive enzymes in the gut of an animal.Examples of such carriers, include plastic capsules or tablets, such asthose known in the art. One method of local administration is by directinjection. Administration of a composition locally within the area of atarget cell refers to injecting the composition centimeters andpreferably, millimeters from the target cell or tissue. The inhibitormay be provided in any suitable form, including without limitation, atablet, a powder, an effervescent tablet, an effervescent powder, acapsule, a liquid, a suspension, a granule or a syrup.

Alternatively, a further embodiment of the invention provides methods ofex vivo cell therapy, wherein a population of pathogenic ABCs isobtained from the subject, contacted, incubated or treated with one ofthe agents disclosed herein for abolishing or decreasing pathogenicABCs, and then administered back to the subject in need thereof.

Obtaining the pathogenic ABCs from the subject would be the same as setforth below for detection of pathogenic ABCs. Administration of the exvivo treated cells of the present invention can be effected using anysuitable route of introduction, such as intravenous, intraperitoneal,intra-gastrointestinal track, subcutaneous, transcutaneous,intramuscular, intracutaneous, intrathecal, epidural, and rectal.According to presently preferred embodiments, the ex vivo treated cellsof the present invention may be introduced to the individual usingintravenous and/or intraperitoneal administration.

Also within the scope of the present disclosure are multipleadministrations (e.g., doses) of the agents and/or populations of cells.In some embodiments, the agents and/or populations of cells areadministered to the subject once. In some embodiments, agents and/orpopulations of cells are administered to the subject more than once(e.g., at least 2, 3, 4, 5, or more times). In some embodiments, theagents and/or populations of cells are administered to the subject at aregular interval, e.g., every six months.

Detection of Pathogenic ABCs

It has also been discovered that pathogenic ABCs, those ABCs that wouldbe found in subjects with or predicted to develop an autoimmune orlymphoproliferative disease, would have different gene expressionprofile than ABCs which are not pathogenic as well as other B cells. Theexpansion and/or function of ABCs that are pathogenic are inhibited bythe SWEF proteins and depend upon IRF5 and other IRFs and these cellsdifferentially express certain genes, including but not limited to,Cxcl9, Cxcl10, Ccl4, Ccl5, Ccl8, Il1r2, Li2rb2, Il18r1, Il18rap, Csf1,Tbx21, Itgax, Itgam, Ctla4, Sema3d, Sema4c, Bmp6, Itga8, Ccl22, Tnsfsf4,Cxcr3, Ccr1, Plxnd1, Itgb1, Ifnγ, Il6, AP1 family members like Jun, Batffamily members, PU.1 and other Ets family members like SpiC, Runx, MyoG,NF-kB, Stat5, Hbp1, Srebf1 and 2, Zbtb32, Nfil3, LifR, Bcl6, Pxk, Smad1,Emp2, Pouf2f2, Rel, Foxj3, Hand1, MafA, MafB, c-Maf, Cebp, Rora, Prdm1,Mertk, Axl, Pparg, CD28, CD9, FcRL5, CD36, CD30/CD30L, c-kit, CD15,CD244, CD68, LXRa, AHR, LDLR, Lmo7, Tnip3, Ppargc1a, Msc, and Mist1.

Most methods start with obtaining a sample of biological tissue or fluidthat contains peripheral blood cells from the subject and extracting,isolating and/or purifying B cells from the tissue or fluid.

Preferred biological tissues include, but are not limited to, epidermal,whole blood, and plasma. The biological tissue obtained from a lymphnode or spleen biopsy.

Preferred fluids include, but are not limited to, plasma, saliva, andurine.

The isolated B cells can be stimulated with an agent including but notlimited to IL21, TLR7 and combinations thereof.

After stimulation nucleic acid is extracted, isolated and purified fromthe cells by methods known in the art.

If required, a nucleic acid sample having the gene sequence(s) areprepared using known techniques. For example, the sample can be treatedto lyse the cells, using known lysis buffers, sonication,electroporation, with purification and amplification occurring asneeded, as will be understood by those in the skilled in the art. Inaddition, the reactions can be accomplished in a variety of ways.Components of the reaction may be added simultaneously, or sequentially,in any order. In addition, the reaction can include a variety of otherreagents which can be useful in the methods and assays and would includebut is not limited to salts, buffers, neutral proteins, such albumin,and detergents, which may be used to facilitate optimal hybridizationand detection, and/or reduce non-specific or background interactions.Also reagents that otherwise improve the efficiency of the assay, suchas protease inhibitors, nuclease inhibitors, and anti-microbial agents,can be used, depending on the sample preparation methods and purity.

Once prepared, mRNA or other nucleic acids are analyzed by methods knownto those of skill in the art. The nucleic acid sequence corresponding toa gene can be any length, with the understanding that longer sequencesare more specific. Preferably a nucleic acid corresponding to a gene isat least 20 nucleotides in length. Preferred ranges are from 20 to 100nucleotides in length, with from 30 to 60 nucleotides being morepreferred, and from 40 to 50 being most preferred.

In addition, when nucleic acids are to be detected preferred methodsutilize cutting or shearing techniques to cut the nucleic acid samplecontaining the target sequence into a size that will facilitate handlingand hybridization to the target. This can be accomplished by shearingthe nucleic acid through mechanical forces, such as sonication, or bycleaving the nucleic acid using restriction endonucleases, or any othermethods known in the art. However, in most cases, the naturaldegradation that occurs during archiving results in “short”oligonucleotides. In general, the methods and assays of the inventioncan be done on oligonucleotides as short as 20-100 base pairs, with from20 to 50 being preferred, and between 40 and 50, including 44, 45, 46,47, 48 and 49 being the most preferred.

A preferred method of the invention is performing gene expressionprofiling of the sample. Gene expression profiling refers to examiningexpression of one or more RNAs in a cell, preferably mRNA. Often atleast or up to 10, 100, 100, 10,000 or more different mRNAs are examinedin a single experiment.

In a preferred method and assay of the invention, the gene expression ofthe mRNA or other nucleic acid obtained from the B cells from thesubject is compared to the reference gene expression of B cells from ahealthy donor. In some cases, these cells are ABCs from a healthy donor,i.e., one without an autoimmune or lymphoproliferative disease. In somecases, the cells are other B cells from a healthy donor.

When the gene expression of the B cells from the subject is beingcompared to ABCs from a healthy donor or control, a finding of at leastone of the following genes being expressed at a higher or greater levelthan the expression in the healthy donor or control would indicate the Bcells from the subject are pathogenic ABCs: Stat5, Hbp1, Sreb1, Zbtb32,Jun, Nfil3, LifR, and AP1-Batf. The expression of any of the geneslisted in Table 1 at a higher or greater level than the expression inthe healthy donor or control would indicate the B cells from the subjectare pathogenic ABCs

When the gene expression of the B cells from the subject is beingcompared to ABCs from a healthy donor or control, a finding of at leastone of the following genes being expressed at a lower level or less thanthe expression in the healthy donor or control would indicate the Bcells from the subject are pathogenic ABCs: MafA, MafB, c-maf, Mertk,Cebp, Rora, PU.1, Mertk, MafB, Spic, Pparg, Ppargc1a, and Prdm1. Theexpression of any of the genes listed in Table 2 at a lower or lesserlevel than the expression in the healthy donor or control would indicatethe B cells from the subject are pathogenic ABCs.

When the gene expression of the B cells from the subject is beingcompared to other B cells a healthy donor or control, a finding of atleast one of the following genes being expressed at a higher or greaterlevel than the expression in the healthy donor or control would indicatethe B cells from the subject are pathogenic ABCs: Cxcl9, Cxcl10, Ccl4,Ccl5, Ccl8, Il1r2, Li2rb2, Il18r1, Il18rap, Csf1, Tbx21, Itgax, Itgam,Ctla4, Sema3d, Sema4c, Bmp6, Itga8, Ccl22, Tnsfsf4, Cxcr3, Ccr1, Plxnd1,Itgb1, Ifnγ, Il6, AP1, Batf, Runx, MyoG, NF-kB, IL-9/IL-9R,IL13/IL-13Ra1, and IL-4.

When the gene expression of the B cells from the subject is beingcompared to other B cells from a healthy donor or control, a finding ofat least one of the following genes being expressed at a lower or lesserlevel than the expression in the healthy donor or control would indicatethe B cells from the subject are pathogenic ABCs: Bcl6, Pxk, Smad1,Emp2, Pouf2f2, PU.1, Rel, Foxj3, and Hand1.

When a method of detecting pathogenic ABCs is used to monitor responseto treatment in a subject, the gene expression from B cells of thesubject after treatment can be compared to the gene expression from Bcells from the subject before treatment. The gene expression from the Bcells of the subject can also be compared to the reference geneexpression of the B cells from a healthy donor or control.

Typically expression is compared to expression of a consistentlyexpressed housekeeping gene transcript, the relative expressiondetermined, and then the expression of the subject is compared to thereference expression of the healthy control:

Methods for examining gene expression, are often hybridization based,and include, Southern blots; Northern blots; dot blots; primerextension; nuclease protection; subtractive hybridization and isolationof non-duplexed molecules using, for example, hydroxyapatite; solutionhybridization; filter hybridization; amplification techniques such asRT-PCR and other PCR-related techniques such as PCR with melting curveanalysis, and PCR with mass spectrometry; fingerprinting, such as withrestriction endonucleases; and the use of structure specificendonucleases. mRNA expression can also be analyzed using massspectrometry techniques (e.g., MALDI or SELDI), liquid chromatography,and capillary gel electrophoresis. Any additional method known in theart can be used to detect the presence or absence of the transcripts.

Alternatively, the level of protein product of the genes can be measuredfrom a protein sample from the biological tissue or fluid using methodsdescribed below.

For a general description of these techniques, see also Sambrook et al.1989; Kriegler 1990; and Ausebel et al. 1990.

The preferred method for the detection of the transcripts is the use ofarrays or microarrays or RNA-sequencing or nanostring.

These terms are used interchangeably and refer to any orderedarrangement on a surface or substrate of different molecules, referredto herein as “probes.” Each different probe of any array is capable ofspecifically recognizing and/or binding to a particular molecule, whichis referred to herein as its “target” in the context of arrays. Examplesof typical target molecules that can be detected using microarraysinclude mRNA transcripts, cRNA molecules, cDNA, PCR products, andproteins.

Microarrays, RNA-sequencing and nanostring are useful for simultaneouslydetecting the presence, absence and quantity of a plurality of differenttarget molecules in a sample. The presence and quantity, or absence, ofthe probe's target molecule in a sample may be readily determined byanalyzing whether and how much of a target has bound to a probe at aparticular location on the surface or substrate.

In a preferred embodiment, arrays used in the present invention are“addressable arrays” where each different probe is associated with aparticular “address.”

The arrays used in the present invention are preferable nucleic acidarrays that comprise a plurality of nucleic acid probes immobilized on asurface or substrate. The different nucleic acid probes arecomplementary to, and therefore can hybridize to, different targetnucleic acid molecules in a sample. Thus, each probe can be used tosimultaneously detect the presence and quantity of a plurality ofdifferent genes, e.g., the presence and abundance of different mRNAmolecules, or of nucleic acid molecules derived therefrom (for example,cDNA or cRNA).

The arrays are preferably reproducible, allowing multiple copies of agiven array to be produced and the results from each easily compared toone another. Preferably microarrays are small, and made from materialsthat are stable under binding conditions. A given binding site or uniqueset of binding sites in the microarray will specifically bind to thetarget. It will be appreciated that when cDNA complementary to the RNAof a cell is made and hybridized to a microarray under suitableconditions, the level or degree of hybridization to the site in thearray corresponding to any particular gene will reflect the prevalencein the cell of mRNA transcribed from that gene. For example, whendetectably labeled (e.g., with a fluorophore) cDNA complementary to thetotal cellular mRNA is hybridized to a microarray, the site on the arraycorresponding to a gene (i.e., capable of specifically binding a nucleicacid product of the gene) that is not transcribed in the cell will havelittle or no signal, while a gene for which mRNA is highly prevalentwill have a relatively strong signal.

By way of example, GeneChip® (Affymetrix, Santa Clara, CA), generatesdata for the assessment of gene expression profiles and other biologicalassays. Oligonucleotide expression arrays simultaneously andquantitatively “interrogate” thousands of mRNA transcripts. Eachtranscript can be represented on a probe array by multiple probe pairsto differentiate among closely related members of gene families. Eachprobe contains millions of copies of a specific oligonucleotide probe,permitting the accurate and sensitive detection of even low-intensitymRNA hybridization patterns. After hybridization data is captured, usinga scanner or optical detection systems, software can be used toautomatically calculate the intensity values for each probe cell. Probecell intensities can be used to calculate an average intensity for eachgene, which correlates with mRNA abundance levels. Expression data canbe quickly sorted based on any analysis parameter and displayed in avariety of graphical formats for any selected subset of genes.

Further examples of microarrays that can be used in the assays andmethods of the invention are microarrays synthesized in accordance withtechniques sometimes referred to as VLSIPS™ (Very Large ScaleImmobilized Polymer Synthesis) technologies as described, for example,in U.S. Pat. Nos. 5,324,633; 5,744,305; 5,451,683; 5,482,867; 5,491,074;5,624,711; 5,795,716; 5,831,070; 5,856,101; 5,858,659; 5,874,219;5,968,740; 5,974,164; 5,981,185; 5,981,956; 6,025,601; 6,033,860;6,090,555; 6,136,269; 6,022,963; 6,083,697; 6,291,183; 6,309,831;6,416,949; 6,428,752 and 6,482,591.

Other exemplary arrays that are useful for use in the invention include,but are not limited to, Sentrix® Array or Sentrix® BeadChip Arrayavailable from Illumina®, Inc. (San Diego, Calif.) or others includingbeads in wells such as those described in U.S. Pat. Nos. 6,266,459;6,355,431; 6,770,441; and 6,859,570. Arrays that have particle on thesurface can also be used and include those described in U.S. Pat. Nos.6,489,606; 7,106,513; 7,126,755; and 7,164,533.

An array of beads in a fluid format, such as a fluid stream of a flowcytometer or similar device, can also be used in methods for theinvention. Exemplary formats that can be used in the invention todistinguish beads in a fluid sample using microfluidic devices aredescribed, for example, in U.S. Pat. No. 6,524,793. Commerciallyavailable fluid formats for distinguishing beads include, for example,those used in XMAP™ technologies from Luminex or MPSS™ methods from LynxTherapeutics.

A spotted microarray can also be used in a method of the invention. Anexemplary spotted microarray is a CodeLink™ Array available fromAmersham Biosciences.

Another microarray that is useful in the invention is one that ismanufactured using inkjet printing methods such as SurePrint™ Technologyavailable from Agilent Technologies. Other microarrays that can be usedin the invention include, without limitation, those described in U.S.Pat. Nos. 5,429,807; 5,436,327; 5,561,071; 5,583,211; 5,658,734;5,837,858; 5,919,523; 6,287,768; 6,287,776; 6,288,220; 6,297,006;6,291,193; and 6,514,751.

DASL can be used for quantitative measurements of RNA target sequencesas well as for DNA target sequences. DASL is described, for example, inFan et al. 2004.

Additional techniques for rapid gene sequencing and analysis of geneexpression include, SAGE (serial analysis of gene expression). For SAGE,a short sequence tag (typically about 10-14 bp) contains sufficientinformation to uniquely identify a transcript. These sequence tags canbe linked together to form long serial molecules that can be cloned andsequenced. Quantitation of the number of times a particular tag isobserved proves the expression level of the corresponding transcript(see, e.g., Velculescu et al. 1995; Velculescu et al. 1997; and de Waardet al. 1999).

Screening and diagnostic method of the current invention may involve theamplification of the target loci. A preferred method for targetamplification of nucleic acid sequences is using polymerases, inparticular polymerase chain reaction (PCR). PCR or otherpolymerase-driven amplification methods obtain millions of copies of therelevant nucleic acid sequences which then can be used as substrates forprobes or sequenced or used in other assays.

Kits

It is contemplated that all of the assays disclosed herein can be in kitform for use by a health care provider and/or a diagnostic laboratory.

Assays for the detection and quantitation of one or more of the genescan be incorporated into kits. Such kits would include probes for one ormore of the genes, reagents for isolating and purifying ABCs and other Bcells and nucleic acids from biological tissue or bodily fluid, reagentsfor performing assays on the isolated and purified nucleic acid,instructions for use, and reference values or the means for obtainingreference values in a control sample for the included genes.

A preferred embodiment of these kits would have the probes attached to asolid state. A most preferred embodiment would have the probes in amicroarray format wherein nucleic acid probes for one or more of thegenes differentially regulated in pathogenic ABCs would be in an orderedarrangement on a surface or substrate.

Drug Screening Assays and Research Tools

All of the biomarkers disclosed herein can be used as the basis for drugscreening assays and research tools.

In one embodiment, IRF5, SWAP-70, and DEF6 polypeptides and proteins canbe used in drug screening assays, free in solution, or affixed to asolid support. All of these forms can be used in binding assays todetermine if agents being tested form complexes with the peptides,proteins or fragments, or if the agent being tested interferes with theformation of a complex between the peptide or protein and a knownligand.

High throughput screening can also be used to screen for therapeuticagents. Small peptides or molecules can be synthesized and bound to asurface and contacted with the polypeptides, and washed. The boundpeptide is visualized and detected by methods known in the art.

Antibodies to the polypeptides can also be used in competitive drugscreening assays. The antibodies compete with the agent being tested forbinding to the polypeptides. The antibodies can be used to find agentsthat have antigenic determinants on the polypeptides, which in turn canbe used to develop monoclonal antibodies that target the active sites ofthe polypeptides.

The invention also provides for polypeptides to be used for rationaldrug design where structural analogs of biologically active polypeptidescan be designed. Such analogs would interfere with the polypeptide invivo, such as by non-productive binding to target. In this approach thethree-dimensional structure of the protein is determined by any methodknown in the art including but not limited to x-ray crystallography, andcomputer modeling. Information can also be obtained using the structureof homologous proteins or target-specific antibodies.

Using these techniques, agents can be designed which act as inhibitorsor antagonists of the polypeptides, or act as decoys, binding to targetmolecules non-productively and blocking binding of the activepolypeptide, or which act as agonists.

A further embodiment of the present invention is gene constructscomprising the genes that encode IRF5, SWAP-70, and Def6 and any of thedifferentially expressed genes described herein, and a vector. Thesegene construct can be used for testing of therapeutic agents as well asbasic research. These gene constructs can also be used to transform hostcells can be transformed by methods known in the art.

The resulting transformed cells can be used for testing for therapeuticagents as well as basic research.

EXAMPLES

The present invention may be better understood by reference to thefollowing non-limiting examples, which are presented in order to morefully illustrate the preferred embodiments of the invention. They shouldin no way be construed to limit the broad scope of the invention.

Example 1 Materials and Methods

Mice

C57BL/6, CD21-Cre and CD11c-Cre were obtained from Jackson Laboratory.DEF6 deficient (Def6^(tr/tr)) mice were generated by LexiconPharmaceuticals, Inc. using a gene trapping strategy as previouslydescribed (Biswas et al. 2012). Swap-70 deficient mice (Swap-70^(−/−))were generated by R. Jessberger as previously described (Biswas et al.2012). Def6^(tr/tr) Swap-70^(−/−) (DKO) mice were generated by crossingDef6^(tr/tr) (Def6ko) mice with Swap-70^(−/−) (Swap70ko) mice that hadbeen backcrossed onto C57BL/6 background for greater than 10 generations(Biswas et al. 2012).

SAP^(−/−) mice were obtained from Taconic and crossed to DKO mice toobtain SAP−/− DKO mice. IL21^(−/−) mice on mixed strain background wereobtained from the Mutant Mouse Regional Resource Centers (Lexicon strainID 011723-UCD), and then backcrossed into a C57BL/6 background forgreater than 10 generations and then crossed with DKO mice to obtainIL21^(−/−) DKO mice.

CD11c CreIRF4^(fl/fl) DKO mice were generated as previously described(Manni et al. 2015).

IRF5^(fl/fl) mice, which do not carry the Dock2 mutation, wereoriginally obtained from Paula Pitha-Rowe (Johns Hopkins University, MD)(Fang et al. 2012). CD21-Cre mice were crossed with IRF5^(fl/fl) mice toproduce CD21-Cre IRF5^(fl/fl) mice. These mice were further crossed withDKO mice expressing either CD21Cre or CD11cCre to produce IRF5^(fl/fl)DKO, CD21-Cre IRF5^(fl/−) DKO, CD11cCre IRF^(fl/−) DKO, and IRF5^(fl/−)DKO mice.

BLIMP-YFP-10BiT double reporter mice have been described previously(Parish et al 2014; Maynard et al. 2007) and were crossed with DKO miceto generate BLIMP-YFP-10BiT DKO mice as described in Chandrasekaran etal. 2016.

Yaa-DKO male mice were obtained by crossing DKO male mice with Yaa micewhich carry a duplication of TLR7 on the Y chromosome (Deane et al.2007).

All mice used in the experiments were kept under specific pathogen-freeconditions. The experimental protocols were approved by theInstitutional Animal Care and Use Committee of the Hospital for SpecialSurgery and WCMC/MSKCC.

Antibodies and Flow Cytometry

The following monoclonal antibodies to mouse proteins were used formultiparameter flow cytometry: CD11c (N418), CD11b (M1/70), CD19 (6D5),B220 (RA3-6B2), T-bet (4B10), CD4 (RM4-5), CD21/CD35 (7E9), CD23 (B3B4),CD86 (GL-1), MHCII (AF6-120.1), IgG1 (RMG1-1) and IgG2a (RMG2a-62) wereobtained from Biolegend. Antibodies to CD43 (S7), CD138 (281-2), GL-7and Fas (Jo2) were obtained from BD. Antibodies to Ki-67 (SolA15), IgD(11-26), IgM (II/41), CD93 (AA4.1), CD5 (53-7.3), PDCA-1 (eBio927), PD1(J43) and Foxp3 (FJK-16s) were obtained from Ebioscience.

For staining of CXCR5 (2G8; BD), cells were incubated in dark at roomtemperature for 25 minutes.

For intracellular staining, cells were fixed after surface staining at4° C. with the Foxp3 Staining Buffer Set (eBioscience) following themanufacturer instruction.

For active caspase-3 staining, cells were stained using the CaspGLOWActive Caspase-3 Staining kit (BioVision) following the manufacturerinstructions. For viability analysis, cells were stained with 0.5 μg ofpropidium iodide/samples prior to acquisition. Data were acquired onFACS Canto (Becton Dickinson) and analyzed with FlowJo (TreeStar)software.

Cell Sorting and B Cell Differentiation

Single-cell suspensions from pooled spleens and lymph nodes werepre-enriched for B cells with B220 microbeads (Miltenyi Biotec)following the manufacturer instructions. B cells were stained with CD11c(N418), CD11b (M1/70), CD19 (6D5), B220 (RA3-6B2) and CD23 (B3B4) andwere sorted on FACS Aria (Becton Dickinson).

B Cell Differentiation

Single-cell suspensions from pooled spleens were enriched for B cellswith biotinylated anti-CD23 (BD Bioscience) and streptavidin microbeads(Miltenyi Biotec) following the manufacturer instructions. CD23+ B cellswere cultured in RPMI 1640 medium (Corning) supplemented with 10% FBS(Atlanta Biologicals), 100 U/ml Penicillin (Corning), 100 mg/mlStreptomycin (Corning), 1× Non-Essential Amino Acids (Corning), 2 mML-Glutamine (Corning), 25 mM Hepes and 50 μM β-Mercaptoethanol, andstimulated with 5 μg/ml F(ab′)2 anti-mouse IgM (αIgM; JacksonImmunoResearch Laboratories), 5 μg/ml Ultra-LEAF purified anti-mouseCD40 (Biolegend), in presence or absence of 50 ng/ml IL-21 (Peprotech),1 μg/ml imiquimod (Invivogen), 10 ng/ml IL-4 (Peprotech) or 20 ng/mlIFN-γ (Peprotech). For proliferation assays, CD23+B cells were labelledwith 2.5 μM CFSE or Cell trace violet (Invitrogen) for 1 minute at roomtemperature prior stimulation.

Real-Time RT-PCR

Total RNA was isolated from cells using RNeasy Plus Mini kit (Qiagen).cDNAs were prepared using the iScript cDNA synthesis kit (Biorad). andanalyzed for the expression of the gene of interest by real-time PCRusing the iTaq Universal SYBR Green Supermix (Biorad). Gene expressionwas calculated using the ΔΔCt method and normalized to Cyclophilin aLifr and Jun primers were obtained from Qiagen.

ccl5 forward (SEQ ID NO: 1) 5′-GCCCACGTCAAGGAGTATTTCTA-3′; ccl5 reverse(SEQ ID NO: 2) 5′-ACACACTTGGCGGTTCCTTC-3′; il6 forward (SEQ ID NO: 3)5′-GAGGATACCACTCCCAACAGAC-3′; il6 reverse (SEQ ID NO: 4)5′-AAGTGCATCATCGTTGTTCATA-3′; cxcl10 forward (SEQ ID NO: 5)5′-CCAAGTGCTGCCGTCATTTTC-3′; cxcl10 reverse (SEQ ID NO: 6)5′-GGCTCGCAGGGATGATTTCAA-3′; ifnγ forward (SEQ ID NO: 7)5′-GGATATCTGGAGGAACTGGC-3′; Ifnγ reverse (SEQ ID NO: 8)5′-GCGCCAAGCATTCAATGAGCTC-3′; spi1 forward (SEQ ID NO: 9)5′-TGCAGCTCTGTGAAGTGGTT-3′; spi1 reverse (SEQ ID NO: 10)5′-AGCGATGGAGAAAGCCATAG-3′; zbtb32 forward (SEQ ID NO: 11)5′-TCCAGATACGGTGCTCCCTTCT-3′; zbtb32 reverse (SEQ ID NO: 12)5′-CCAGAGAGCTTTGGAGTGGTTC-3′; Nfil3 forward (SEQ ID NO: 13)5′-AATTCATTCCGGACGAGAAG-3′; Nfil3 reverse (SEQ ID NO: 14)5′-CGATCAGCTTGTTCTCCAAA-3′; Maf forward (SEQ ID NO: 15)5′-AGCAGTTGGTGACCATGTCG-3′; maf reverse (SEQ ID NO: 16)5′-TGGAGATCTCCTGCTTGAGG-3′; axl forward (SEQ ID NO: 17)5′-CGAGAGGTGACCTTGGAAC-3′;DNA Constructs

Expression plasmids for untagged and HA-tagged DEF6 were generated asdescribed previously (Biswas et al. 2012). The full-length wild typehuman SWAP-70 expression plasmid (pIRES2-EGFP-HA-SWAP70) was constructedby cloning the entire coding region of the human Swap-70 cDNA, fused inframe with a hemagglutinin (HA) epitope coding sequence at its 5′ end,into the pIRES2-EGFP bicistronic expression vector (Clonetech). Variousdeletion mutants of human SWAP-70 were generated by PCR usingappropriate primers. The full-length wild type human IRF5 expressionconstruct in pcDNA3 was a kind gift of Dr. Inez Rogatsky. Full lengthhuman IRF5 (variant 5) and T-bet expression constructs were purchasedfrom Genescript. Expression plasmids for Flag-tagged IRF5 (variant 5)and its various deletion mutants were constructed in p3XFLAG-CMV-10expression vector (Sigma) using IRF5 construct (Genescript) as atemplate. Expression plasmid for untagged T-bet was generated inpIRES2-EGFP bicistronic expression vector (Clonetech) using T-betexpression construct (Genescript) as a PCR template.

Western Blotting and Immunoprecipitation

Nuclear and cytoplasmic extracts were prepared with NEPER Nuclear andCytoplasmic Extraction Reagents (Pierce), as previously described(Biswas et al. 2012). For expression analysis cell extracts wereanalyzed by Western blotting with anti-STAT3 (BD Bioscience),anti-pSTAT3 (Y705) (Cell Signaling), anti-IRF5(Cell Signaling) oranti-HDAC1 (Cell Signaling) antibodies. For protein-proteininteractionstudies, cell extracts were immunoprecipitated with ananti-IRF5 (Cell Signaling), or anti-HA (3F10; Roche Applied Science)antibodies. The immunoprecipitates were resolved by 8% SDS-PAGE,transferred to a nitrocellulose membrane, and then immunoblotting witheither an anti-SWAP-70 antibody (Santa Cruz Biotechnology, Inc.),anti-DEF6 antiserum (Gupta et al. 2003) or anti-HA antibody (RocheApplied Science).

ChIP Assays

CD23+ B cells were purified and stimulated in vitro for 48 hours. Afterharvesting, the cells were cross-linked with formaldehyde, and chromatinextracts were prepared using the truChIP Chromatin Shearing Reagent Kit(Covaris) according to manufacturer instructions. The DNA-proteincomplexes were immunoprecipitated with an anti-IRF5 (Abcam, ab21689) oranti-T-bet (Santa Cruz; sc-21749X) specific antibody or a controlantibody. After cross-linking was reversed and proteins were digested,the DNA was purified from the immunoprecipitates as well as from inputextracts, and then analyzed by quantitative PCR using the followingprimers within the ABC-specific ATAC-seq peaks (murine):

Il6 TSS (Forward): (SEQ ID NO: 18) 5′-AGCTTCTCTTTCTCCTTATAAAACATTG-3′;Il6 TSS (Reverse); (SEQ ID NO: 19) 5′-GCATCGAAAGAATCACAACTAGG-3′;Cxcl10 Cluster (Forward):  (SEQ ID NO: 20) 5′-AGTAGTCCCCACTGTCTGACT-3′;Cxcl10 Cluster (Reverse):  (SEQ ID NO: 21) 5′-GTGAGTCCCTTTAGCACCAGA-3′;Zeb2 Exon8 (Forward): (SEQ ID NO: 22) 5′-AGCAGTCCCTTTATGAACGG-3′;Zeb2 Exon8 (Reverse): (SEQ ID NO: 23) 5′-GCTTCCATCCCTACACCTAAG-3′;Jun (Forward): (SEQ ID NO: 24) 5′-AGAACAGCTTTTGAGCACCG-3′;Jun (Reverse): (SEQ ID NO: 25) 5′-TGGCTTCAAAGTGACTAACAGCA-3′;IgG2c (Forward): (SEQ ID NO: 26) 5′-TGTAATGCCTGGTTGCCTCC-3′;IgG2c (Reverse) (SEQ ID NO: 27) 5′-GTTCGGGACCCACAGTACATT-3.ONP Assays

ONP assays were conducted as previously described (Biswas et al. 2010).Briefly, nuclear extracts were precleared with streptavidin-agarosebeads and then incubated with trimerized biotinylated double-strandedoligonucleotide containing potential IRF binding site within theATAC-seq peak at the IL-6 TSS (5′-TGCTGAGTCACTTTTAAAGAAAAAAAGAAGAGT-3′)(SEQ ID NO: 28) or the CXCL10 Cl(5′-CATAGAAAATGTTTTCAAAACCCGCATTCCGCTTATGCTGTCTGGTATCTGAAATAGATCTGTCAGGGGGTCACATTTTATAAGCACCACTTCGTGTTTG-3′)(SEQ ID NO: 29). Proteins bound to the biotin-labeled DNA were collectedby streptavidin-agarose beads, separated by 8% SDS-PAGE, and analyzed byWestern blotting using anti-mouse IRF5 Ab (Cell signaling), anti-humanIRF5 Ab (Santa Cruz SC-390364) or an anti-T-bet Ab (Santa Cruz;sc-21749).

Cytokines ELISA

IL-6 and CXCL10 in culture supernatants were measured using the mouseELISA Max Standard Set (Biolegend) and the mouse Quantikine ELISA kit(R&D Systems) respectively.

Anti-ds DNA ELISA and ANA

For anti-dsDNA ELISA, plates were coated with 100 μg/ml salmon sperm DNA(Invitrogen AM9680) at 37° C. overnight and blocked in 2% BSA in PBS, atroom temperature for 2 hours. For anti-cardiolipin ELISA Immulon 2HBplates (ThermoFisher) were coated with 75 μg/ml of cardiolipin dissolvedin 100% ethanol at room temperature overnight. Sera were diluted 1:200and incubated on coated plates at room temperature for 2 hours. Plateswere then incubated with horseradish peroxidase-labelled goat anti-mouseIgG, IgG1 or IgG2 Fc antibody for 1 hour (eBioscience). Anti-ssDNA andanti-nRNP IgG ELISAs were obtained from Alpha Diagnostic International.OD₄₅₀ was measured on a microplate reader. ANAs were detected on Hep-2slides (MBL international) at a 1:200 dilution using Alexa Flour488-conjugated anti-mouse IgG (Jackson ImmunoResearch). Fluorescentintensity was semi-quantitated by following the guidelines establishedby the Center for Disease Control, Atlanta, Georgia

Histology and Immunofluorescence Staining

Tissue specimens were fixed in 10% neutral buffered formalin andembedded in paraffin. Tissue sections were stained with periodic acidschiff (PAS) and analyzed by light microscopy. The nephritis scoringsystem was adapted from the International Society of Nephrology/RenalPathology Society (ISN/RPS) classification of human lupus nephritis. Atleast 40 glomeruli per mouse were evaluated. The final score accountedfor morphological pattern (mesangial, capillary, membranous) and for thepercentage of involved glomeruli. Immunofluorescence analysis on frozenkidney sections was performed by staining with FITC-labeled goatanti-mouse IgG (Jackson ImmunoResearch Laboratories) and specimens wereanalyzed with a LSM 510 laser scanning confocal microscope (Carl Zeiss,Inc.). Images were captured by Q capture software. Five representativeglomeruli per mouse were chosen and mean fluorescent intensity (MFI) wascalculated using ImageJ software.

RNA Seq Analysis

Total RNA was isolated using RNeasy Plus Mini kit (Qiagen). SMARTSeq v3Ultra Low Input RNA Kit (Clontech) followed by Nextera librarypreparation were used to prepare Illumina-compatible sequencinglibraries. Quality of all RNA and library preparations were evaluatedwith BioAnalyser 2100 (Agilent). Sequencing libraries were pair-endsequenced by the Weill Cornell Epigenomics Core using HiSeq2500 at thedepth of ˜30-50 million fragments per sample. Sequencing performance wasevaluated using FASTQC. 50-bp paired reads were mapped to mouse genome(mm10, build 38.75, 41,128 genes and 87,108 transcripts) with CLC BioGenomic Workbench 7.5 software (Qiagen).

Duplicated reads with more than 5 copies were discarded. Read counttables were created using unique exon read counts and the differentialexpression was analyzed using EDGER (Bioconductor). Genes with theexpression levels less than 1 cpm in at least three conditions wereconsidered non-expressing and removed from further analysis. A negativebinomial generalized log-linear model was fit to read counts for eachgene. A likelihood ratio tests with the null hypothesis that thepairwise contrasts of the coefficients are equal to zero was used toevaluate the significance of differences in expression between analyzedgroups. Benjamini-Hochberg false discovery rate (FDR) procedure was usedto correct for multiple testing. Genes with a FDR-corrected p-value>0.01and less than 2 fold change were filtered out. Genes that passed thefiltering were considered to be differentially expressed. Gene SetEnrichment Analysis was performed using the difference oflog-transformed count per million (cpm) for contrasted conditions as aranking metric. Molecular Signatures DataBase v 5.2 (Broad Institute)was used as source of gene sets with defined functional relevance. Genesets ranging between 15 and 1000 genes were included into analysis.Nominal p values were FDR corrected and gene sets with FDR<0.05 wereused to create GSEA enrichment plot. To define the groups of potentiallyco-regulated genes, unsupervised hierarchical clustering analysis oflog-transformed expression values (cpm) in R was performed. Thedistances between genes were calculated as (1−Pearson correlation). TheEuclidean distance was used to determine the distances between samples.Ward.D2 methods was used to performs clustering. The expression valueswere z-transformed and visualized using heatmaps.

ATAC-seq, Peak Calling and Annotation

The nuclei of sorted WT and DKO ABC or DKO Follicular B cells wereprepared by incubation of cells with nuclear preparation buffer (0.30 Msucrose, 10 mM Tris, pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mMEGTA, 0.1% NP40, 0.15 mM spermine, 0.5 mM spermidine and 2 mM 6AA)(Minnich et al. 2016). Libraries were prepared as described previously(Buenrosto et al. 2015). Paired-end 50 bp sequences were generated fromsamples on an Illumina HiSeq2500. The makeTagDirectory was used followedby findPeaks command from HOMER version 4.7.2 to identify peaks ofATAC-seq. A false discovery rate (FDR) threshold of 0.001 was used forall data sets. The following HOMER command was used: cmd=findPeaks<sample tag directory>-style factor or histone-o<output file>-i<inputtag directory>. The total number of mapped reads in each sample wasnormalized to ten million mapped reads. Peak-associated genes weredefined based on the closest genes to these genomic regions using RefSeqcoordinates of genes. The annotatePeaks command from HOMER was used tocalculate ATAC-seq tag densities from different experiments and tocreate heatmaps of tag densities. Sequencing data were visualized bypreparing custom tracks for the UCSC Genome browser.

Motif Enrichment Analysis

De novo transcription factor motif analysis was performed with motiffinder program findMotifsGenome from HOMER package, on given ATAC-seqpeaks. Peak sequences were compared to random genomic fragments of thesame size and normalized G+C content to identify motifs enriched in thetargeted sequences.

Statistics

P values were calculated with unpaired two-tailed Student's t-test fortwo-group comparisons and by one-way ANOVA followed by Bonferroni'smultiple comparisons test for multi-group comparisons. For statisticalanalysis of ANA intensity score the non-parametric Mann-Whitney test wasused. P values of <0.05 were considered significant. Ns: notsignificant, *: p≤0.05, **: p≤0.01 ***: p≤0.001***: p≤0.0001.Statistical analysis was performed with Graphpad Prism 5.

Example 2 Female DKO Mice Have Premature Expansion of Pathogenic ABCs,which is Dependent on the Absence of Both SWEF Proteins and Contributeto Lupus

The finding that ABCs expand in autoimmune mouse strains coupled withthe spontaneous development of autoimmunity in DKO mice prompted theinvestigation as to whether this B cell subset accumulates prematurelyin these mice. The mice and methods described in Example 1 were used.

As compared to wild type mice, DKO female mice demonstrated a markedincrease in the frequencies and numbers of splenic B cells expressingCD11c and CD11b (FIG. 1A). This increase could be observed by gatingeither on B220 alone or on both B220 and CD19 (FIG. 1A). Expansion ofCD11c+CD11b+ B cells was primarily observed in spleens and, onlyminimally, in lymph nodes (FIG. 1B). Further staining for PDCA-1confirmed that accumulation of these cells was not due to an increase inplasmacytoid dendritic cells (results not shown).

While ABCs in wild type female mice are normally detected after 12months of age, ABCs in DKO female mice started appearing by 10 weeks ofage (FIG. 1C), were readily observed by 18 weeks of age (FIG. 1D), andcomprised up to 15% of splenic B cells in older (>23 weeks) mice (FIG.1A).

Since B cells express both DEF6 and SWAP-70, it was also examinedwhether lack of either DEF6 alone or SWAP-70 alone was sufficient topromote the accumulation of ABCs in vivo (FIG. 1B). While a smallincrease in the frequencies of these cells could be observed in thespleens of female mice lacking only DEF6 or only SWAP-70, theirabundance did not reach the levels observed in DKO female mice (FIG.1B). The premature expansion of CD11c+CD11b+ B cells observed in DKOmice was thus dependent on the concomitant absence of DEF6 and SWAP-70,thus, both SWEF proteins control the accumulation of these cells invivo.

To further characterize the CD11c+CD11b+ B cells that accumulated in DKOfemale mice, the expression of several markers whose presence or absencedefines ABCs was examined (FIG. 1E). As expected, the expression ofTbet, a major regulator of ABC generation, was significantly higher inCD11c+CD11b+ DKO B cells as compared to CD11c−CD11b− DKO B cells andcorresponded to a marked expansion of CD11c+T-bet+ B cells in DKO femalemice. Moreover, CD11c+CD11b+ DKO B cells downregulated the expression ofCD21 and CD23 and expressed high levels of CD86, MHCII, and IgM (FIG.1D). CD11c+CD11b+ DKO B cells did not express CD5, CD43, or CD93 (FIG.1D). While most CD11c+CD11b+ DKO B cells expressed sIgM, a small numberof these cells had undergone class-switching and expressed IgG1 andIgG2c (FIG. 1F).

In order to investigate whether ABCs contribute to the development oflupus in DKO female mice, their ability to produce autoantibodies wasinvestigated. CD11c+CD11b+ B cells from DKO mice were FACS-sorted andcultured in vitro in the presence or absence of the TLR7 agonist,imiquimod (FIG. 1G). ABCs, but not Follicular B cells (FoB) from DKOmice secreted anti-dsDNA IgG2c (FIG. 1G). No anti-dsDNA IgG1 productionwas instead observed under these stimulatory conditions (not shown).TLR7 stimulation of DKO ABCs also resulted in the production ofanti-nRNP and anti-cardiolipin IgG antibodies (FIG. 1G). DKO ABCs canthus directly contribute to the autoimmune syndrome in DKO mice byproducing autoantibodies and are thus denoted as “pathogenic ABCs”.

Example 3 IL-21 Regulates the Generation of ABCs in DKO Mice In Vitroand In Vivo

In addition to TLRs, T cells can also promote the generation of ABCs byproviding contact-dependent signals and cytokines like IL-21 (Naradikianet al. 2016). An in vitro system to directly investigate the ability ofthese signals to drive the formation of ABCs from B cells purified from8 week-old wild type or DKO female mice (Example 1) was set up (FIG.2A). Culturing wild type or DKO B cells with αIgM (5 μ/ml) and αCD40 (5μg/ml), either alone or with imiquimod, (1 μg/ml) did not result in theformation of CD11c+CD11b+ B cells. However, the addition of IL-21 (50ng/ml) led to a significantly greater population of CD11c+CD11b+ cellsin cultures of DKO than wild type B cells (FIG. 2A). A population ofCD11c+CD11b− B cells could also be observed in these DKO cultures (FIG.2A). Similar results were obtained by using either CD11c and T-bet orCD11c and CD11b as markers (FIGS. 2A and 2B). In line with previousreports (Naradkian et al. 2016a), stimulation of wild type and DKO Bcells with either IL-4 or IFNγ alone did not result in the formation ofCD11c+T-bet+ B cells and addition of IL-4 inhibited the IL-21-mediatedformation of these cells in both wild type and DKO cultures (results notshown). DKO B cells therefore exhibited an increased ability to generateABCs in vitro in response to IL-21 stimulation.

To further evaluate the importance of IL-21 in the aberrant expansion ofABCs observed in DKO mice, DKO female mice that also lack IL-21 (IL-21koDKO) were generated. Accumulation of CD11c+CD11b+ B cells was completelyabrogated in these mice as compared to age-matched female DKO mice (FIG.2C). DKO mice lacking IL-21 also failed to accumulate T_(FH) cells,germinal center (GC) B cells, and plasma cells, and failed to produceanti-dsDNA autoantibodies (FIGS. 2E and 2F).

To determine whether, in addition to IL-21 production, direct T-Binteractions were also necessary for the expansion of DKO ABCs in vivo,their presence in DKO mice that lack SAP (SLAM-associated protein) wasexamined. SAP is required to mediate sustained interactions between Tand B cells (Fang et al. 2012). As shown in FIG. 2D, the concomitantabsence of SAP in DKO mice (SAPko DKO) resulted in diminishedaccumulation of pathogenic ABCs. This was again accompanied by profoundreductions in T_(FH) cells, GC B cells, plasma cells and markedly loweranti-dsDNA autoantibody titers (FIGS. 2E and 2F). Thus, the aberrantexpansion of pathogenic ABCs observed in DKO mice is dependent on IL-21and cognate T-B cell interactions, signals that also control thedevelopment of lupus.

Example 4 The SWEF Proteins Regulate the Proliferation andProinflammatory Capacity of ABCs

B cells were sorted based on the levels of expression of CD11c and CD11band RNA-Seq employed as described in Example 1 to compare thetranscriptome of CD11c+CD11b+ (ABC) DKO B cells to that of CD11c−CD11b−(FoB) B cells obtained from either wild type or DKO mice. A total of3049 genes were differentially expressed amongst the three differentsubsets (FIG. 3A). A set of genes (cluster 2, DKO-specific up) wasupregulated or downregulated (cluster 1, DKO-specific down) in DKO Bcells irrespective of the expression of CD11c and CD11b suggesting thatthe lack of SWEF proteins altered the expression of these genes in Bcells independently of their differentiation state (FIG. 3A).

To gain insights into the critical processes controlled by the SWEFproteins in B cells, the transcriptional profile of FoBs from wild typemice was first compared to that of FoBs from DKO mice. Based on gene setenrichment analysis (GSEA) (FIG. 3B) lack of the SWEF proteins affectedthe control of B cell proliferation, potentially, via E2F family oftranscription factors and regulators of the G2/M checkpoint such as Wee1and Chek1.

To extend and confirm these observations, the proliferative capabilitiesof B cells in wild type and DKO mice were assessed by staining for Ki67.As compared to wild type B cells, CD11c−Tbet− DKO B cells contained asmall population of highly proliferative cells (FIG. 3C). Strikingly,CD11c+Tbet+ DKO B cells proliferated even more robustly than CD11c−Tbet−DKO B cells (FIG. 3C).

No differences in apoptotic rates were instead observed between wildtype and DKO B cells (FIG. 3D). In vitro experiments wherein CD23+ Bcells were purified from wild type and DKO female mice (6-9 weeks old),labeled with CFSE and cultured with αIgM (5 μg/ml), αCD40 (5 μg/ml), andIL-21 (50 ng/ml) for 3 days, demonstrated that DKO ABCs proliferated toa greater extent than WT ABCs upon stimulation with IL-21 (FIG. 3E). Inline with the in vivo findings, WT and DKO B cells exhibited similarsurvival rates in vitro as assessed by either PI staining or Caspase 3cleavage at different times after stimulation with αIgM (5 μg/ml), αCD40(5 μg/ml), IL-21c (50 ng/ml) or imiquimod (1 μg/ml) for 3 or 5 days(FIG. 3F). Thus, SWEF proteins regulate the proliferation of B cells,and play a particularly important role in restraining the capacity ofpathogenic ABCs to proliferate in response to IL-21.

In addition to clusters 1 and 2 that were differentially expressed inall DKO B cells, the transcriptomic analysis also uncovered additionalclusters of genes (clusters 3 and 5), which were specificallyupregulated in CD11c+CD11b+ DKO B cells as compared to CD11c−CD11b− Bcells obtained from either wild type or DKO mice (FIGS. 3A and 3G). Asexpected, DKO ABCs expressed higher levels of T-bet (Tbx21), CD11c(Itgax) and CD11b (Itgam), which were used for ABC isolation, ascompared to FoBs (FIG. 3G).

GSEA was used to gain insights into the pathways that were uniquelyupregulated in DKO ABC cells. Notably the top enriched sets (FDR,q<0.05) included several gene sets enriched in transcripts controllingchemotaxis, integrin binding, cell adhesion, and inflammation (FIGS. 3Hand 3J). Prominent amongst the upregulated genes were a number ofchemokines (e.g. Cxcl9, Cxcl10, Ccl4, Ccl5, Ccl8), cytokine receptors(Il1r2, Il12rb2, Il18r1, and Il18rap) and cytokines including Csf1(FIGS. 3G and 3K), some of which were further validated by qPCR in FoBand ABC populations sorted from WT and DKO female mice (FIGS. 3I and3J). Thus, as compared to FoBs, the ABCs from DKO female mice wereendowed with proinflammatory capabilities and unique migratory andadhesive attributes. In addition to promoting the proliferation ofpathogenic ABC cells, the lack of SWEF proteins altered theirmigratory/adhesive attributes and endowed them with enhancedproinflammatory functions.

Example 5 The Chromatin Landscape of DKO ABC Cells is Enriched with IRFand AP-1/BATF Motifs as Compared to FoB Celle

The distinctive transcriptional program of DKO ABC cells suggested thatthese cells might exhibit a unique chromatin landscape. To directlyaddress this possibility, ATAC-seq (assay for transposase-accessiblechromatin using sequencing), which enables the identification ofaccessible regions of chromatin even in small number of cells asdescribed in Example 1 and Buenrostro et al. 2015 was employed.

ATAC-seq signals from sorted CD11c+CD11b+ DKO cells were compared tothose from sorted CD11c−CD11b− cells from the same mice. The focus ofthe analysis was on regions with higher signals and 3,666 ABC-specificpeaks that satisfied those criteria were identified (FIG. 4A). The ABCspecific peaks were primarily found in intergenic (45%) and intronic(50%) regions and only rarely in promoters. Loci which weredifferentially accessible in ABCs as compared to CD11c−CD11b− cellsincluded a number of proinflammatory cytokines like IFNγ and IL-6 andother ABC-specific targets like the CXCL10 cluster of genes (FIG. 4B).Consistent with the results of the transcriptomic analysis, ABC-specificpeaks were positively associated with transcriptionally active genes inABC DKO cells as compared to FoB DKO cells and pathway analysis showedthat many of the differentially expressed ATAC-seq associated genes wereinvolved in locomotion and cellular adhesion (Table 5 and FIG. 4D).

To gain insights into the molecular mechanisms responsible for thedistinctive chromatin profile of DKO ABCs, the transcription factormotifs enriched in ABC specific peaks were determined (Tables 3 and 4).ABC-specific accessible loci displayed enrichment in AP-1/BATF, IRF, andT-bet binding motifs (Table 3). Interestingly, the ABC specific peaksexhibited substantial positional bias in the distribution of IRF andT-bet binding motifs, which coincided with the peak summit (FIG. 4C).This pattern contrasted with that observed in FoB-specific peaks, whichexhibited enrichment in motifs for a distinct set of transcriptionfactors including POU2F2 (Tables 3 and 4, FIG. 4C). Thus, DKO ABCs, ABCsthat aberrantly expand in this autoimmune setting, i.e., pathogenicABCs, exhibited a unique chromatin landscape, which, in addition toT-bet motifs, is enriched in IRF and AP-1/BATF motifs and correlateswith a distinctive transcriptional profile.

TABLE 3 Best Match for Motifs in DKO ABC-Specific Peaks Best MatchP-value AP1-BATF 1e−116 IRF 1e−107 T-bet 1e−102 PU.1 1e−73  RUNX 1e−72 MyoG 1e−45  NF-κB 1e−28 

TABLE 4 Best Match for Motifs for DKO FoB-Specific Peaks Best MatchP-value POU2F2 1e−81 E2A-PU.1 1e−41 REL 1e−14 Foxj3 1e−14 Hand1 1e−12

TABLE 5 Functionally Enriched Gene Ontology (GO) Categories of Genesassociated with ABC-Specific Peaks of ATAC-seq (n = 487). FDRDescription P-value q-value GO_IMMUNE_SYSTEM PROCESS 4.17E−27 1.85E−23GO_POSTIVE_REGULATION_OF_ 1.02E−22 2.25E−19 RESPONSE_TO_ STIMULUSGO_LOCOMOTION 3.04E−21 4.28E−18 GO_CELLULAR_ 3.86E−21 4.28E−18RESPONSE_TO_ORGANIC_SUBSTANCE GO_BIOLOGICAL_ADHESION 1.40E−20 1.25E−17

Example 6 Distinctive Transcriptional and Epigenomic Programs ofAutoimmune Prone DKO ABCs as Compared to Non-Autoimmune ABCs

To gain insights into the programs that are specifically dysregulated inpathogenic, autoimmune-prone DKO ABCs as compared to the ABCs thatslowly accumulate in non-autoimmune WT female mice, CD11c+CD11b+ B cellsfrom older WT female mice were sorted and compared their transcriptionalprofiles to those of CD11c+CD11b+ B cells derived from DKO female mice.

WT and DKO ABCs expressed similar levels of T-bet (Tbx21) (FIG. 5A). Atotal of 711 genes were differentially expressed between the twopopulations, of which 111 genes were upregulated in DKO ABCs as comparedto WT ABCs and 600 genes were downregulated (Tables 1 and 2 and FIG.5B). Notably, ABCs from DKO mice expressed higher levels ofimmunoglobulin gene transcripts than ABCs derived from WT mice butdownregulated a subset of myeloid-related transcripts (FIGS. 5B and 5C).The increased levels of immunoglobulin gene transcripts displayed by theDKO ABCs were not associated with changes in the expression of keyregulators of plasma cell differentiation like IRF4, IRF8, Bcl6 orBlimp1 (FIG. 5A) suggesting that the differences between WT and DKO ABCswere not due to the presence of contaminating plasmablasts within theDKO ABC population.

DKO ABCs, however, exhibited selective changes in the expression ofother transcription factors including upregulation of Jun and NFIL-3 anddownregulation of c-maf, MafB, and PPAR-γ while levels of PU.1 weresimilar to those of WT ABCs (FIG. 5A). Differential expression ofselected targets, which included key regulators of apoptotic cellengulfment like Mertk and Axl, was further confirmed by qPCR (FIG. 5D).Thus, the ABCs that accumulate in autoimmune-prone DKO female mice wereendowed with a higher immunoglobulin producing capacity than WT ABCs butdownregulate some of the myeloid related features that can be associatedwith this B cell subset. Also as shown by qPCR, MAFB, NDNF and Thsd7awere differentially expressed between wild type ABCs and DKO ABCs (FIG.5E).

To investigate the differences in the chromatin landscape of WT and DKOABCs that might accompany these distinct transcriptional profiles,ATAC-seq signals from sorted WT ABCs were compared to those of DKO ABCs.27,483 WT ABC-specific peaks and 1,583 DKO-ABC specific peaks (FIG. 5F)were identified. Most of the WT or DKO ABC-specific peaks were primarilyfound in intergenic and intronic regions and only rarely in promoters,with 44% being intergenic and 46% being intronic regions in the WT-ABCspecific peaks (n=27,483) and 51% being intergenic and 43% beingintronic regions of DK-ABC specific peaks (n=1,583). DKO ABC-specificaccessible loci again displayed enrichment in IRF, AP-1/BATF, and T-betbinding motifs (Table 6). In contrast, the pattern associated with WTABC-specific peaks was associated with enrichment in PU.1, MAF, andC/EBP binding motifs (Table 7). These results were consistent with thedownregulation of MAF and MAF-B observed in DKO ABCs and were reflectedin differences in the accessibility of the MAF and MAF-B loci in theATAC-seq (results not shown). In line with the results of thetranscriptomic analysis and supporting the idea that the differentialchromatin accessibility between WT and DKO ABCs is functionallyimportant, gene ontology (GO) categories of genes associated withWT-specific or DKO-specific peaks of ATAC-seq indicated that WTABC-specific peaks were positively associated with transcriptionalprograms regulating phagocytosis and other myeloid-related functionswhile DKO ABC-specific peaks were enriched in processes linked to B celldifferentiation, activation and Ig regulation (FIG. 5G).

Thus, in comparison to the ABCs that slowly accumulate in WT mice withage, i.e., non-pathogenic ABCs, the chromatin landscape of pathogenicautoimmune-prone ABCs was characterized by dual abnormalities wherebyenrichment in IRF and AP-1/BATF motifs was coupled with depletion ofPU.1- and MAF-bound regulatory regions.

TABLE 6 Best Matches to Motifs in DKO ABC-Specific ATAC-seq Peaks BestMatch P-value IRF 1e−81 AP1-BATF 1e−29 STAT5 1e−28 Hbp1 1e−27 SREBF11e−25 Tbet 1e−24

TABLE 7 Best Matches to Motifs in WT ABC-Specific ATAC-seq Peaks BestMatch P-value PU.1 1e−1151 MafA 1e−165  CEBP 1e−109  RORA 1e−107  PRDM11e−84   PU.1-RF 1e−68  

Example 7 Expansion of ABCs Depends Upon IRF5

While ABC generation is known to be dependent on Tbet (Rubtsova et al.2015; Naradikian et al. 2016), the role of the IRFs in the formation andfunction of ABCs is unknown. Given that the SWEF proteins can regulatethe activity of IRF4 (Biswas et al. 2012; Manni et al. 2015), ananalysis of whether the aberrant expansion of ABCs in DKO mice mightdepend on this transcription factor was performed, using CD11c-CreIRF4fl/flDKO mice (Manni et al. 2015.) However, it was determined thatdeleting IRF4 in CD11c+ expressing cells did not affect the accumulationof ABCs (results not shown) or any of the autoimmune parameters thatcharacterize the development of lupus in DKO mice (Manni et al. 2015).Thus, the dysregulated expansion of ABCs in DKO mice does not rely onIRF4.

Given the high degree of homology amongst IRF DNA binding domains thepossibility that another IRF may regulate the aberrant generation of DKOABCs was investigated. The focus was on IRF5 given its ability toregulate the production of IgG2a/c, proinflammatory mediators like IL-6and the strong association with SLE (Cham et al. 2012; Lazzari andJefferies 2014). To facilitate these studies, DKO mice completelylacking IRF5 in B cells (CD21Cre-IRF5^(fl/−) DKO) were generated andthen assessed the ability of B cells from these mice to generate ABCs invitro. Expression of IRF5 was similar in WT and DKO B cells and wasabsent in B cells from CD21Cre-IRF^(5fl/−) DKO mice (results not shown).

Lack of IRF5 markedly diminished the ability of DKO B cells to generateABCs in cultures supplemented with IL-21 (FIGS. 6A and 6B). Theincreased ability of DKO B cells to produce IL-6 and CXCL10 upon IL-21stimulation was also decreased by the absence of IRF5 (FIGS. 6C and 6D).Deleting IRF5 in DKO B cells also profoundly decreased theIL-21-mediated production of IgG2c, but not that of IgG1 (FIG. 6E).Expression of Jun was also dysregulated in DKO B cells in an IL-21- andIRF-5-dependent manner (FIG. 6F). Thus, the IL-21 driven abnormalitiesin ABC generation and function exhibited by DKO B cells are dependent onthe presence of IRF5.

Given that the ATAC-seq analysis had revealed an enrichment of IRFbinding sites in ABC specific peaks located at the Il6 TSS, the Cxcl10cluster, the IgG2c region, and Jun, ChIP-assays were performed to assessthe binding of IRF5 to these regulatory regions. As compared to wildtype B cells, DKO B cells exhibited enhanced binding of IRF5 to thesesites upon IL-21 stimulation despite exhibiting similar levels of Stat3phosphorylation and IRF5 nuclear translocation (FIGS. 6G and 6H).Minimal IRF5 binding was observed in IRF5-deleted DKO B cells or whencells were stimulated in the absence of IL-21 supporting the specificityof the findings (FIG. 6G).

To evaluate whether ABC-specific peaks bound by IRF5 could also betargeted by T-bet, ChIP-assays with a T-bet antibody were performed(FIG. 6I). DKO B cells exhibited increased binding of T-bet to theABC-specific region at the CXCL10 cluster, the IgG2c peak, and Jun butnot to the IL-6 TSS or the negative control, a site in Zeb2 genepreviously shown not to bind T-bet (Dominguez et al. 2015). Notably,IRF5 deletion in DKO B cells resulted in decreased binding of T-bet tothe CXCL10 cluster, the IgG2c peak, and Jun. Further corroboration thatIL-21 stimulation of DKO B cells leads to an aberrant ability of IRF5and T-bet to target the CXCL10 cluster was obtained by performingoligonucleotide precipitation assays (ONPs). As observed with the ChIPassays, the presence of IRF5 was necessary for the ability of T-bet tobind to the CXCL10 cluster while no binding of T-bet to the IL-6 TSScould be detected (FIG. 6J). Co-transfection of T-bet with IRF5 coupledwith a mutational analysis further confirmed that optimal recruitment ofT-bet to the CXCL10 cluster requires DNA binding by IRF5 (FIG. 6K) Takentogether these findings support a model whereby, in the absence of SWEFproteins, IL-21 stimulation leads to an increased ability of IRF5 totarget ABC-specific peaks. Targeting of these regions by IRF5subsequently enables strong recruitment of T-bet to a subset of thesesites.

To delineate the mechanisms by which the lack of SWEF proteins resultsin enhanced IRF5 activity, the possibility that they can directlyinteract with IRF5 and thus restrain its activity was investigated. Asshown in FIG. 6L, coimmunoprecipitation experiments indeed revealed thatendogenous IRF5 in B cells interacts with both DEF6 and SWAP-70. Amutational analysis furthermore revealed that the association of IRF5with either DEF6 or SWAP-70 maps to the C-terminal portion of the SWEFproteins, which contains their IRF-interacting region, and requires theIAD (IRF Association Domain) of IRF5, a domain within IRFs known tomediate protein-protein interaction (results not shown). No interactionof either DEF6 or SWAP-70 with T-bet could instead be detected (FIG.6M). Co-transfections of IRF5 with DEF6 or SWAP-70 followed by ONPassays demonstrated that the full-length SWEF proteins, but not mutantsof these molecules which are unable to interact with IRF5, interferewith the ability of IRF5 to bind to the IL-6 TSS (FIG. 6N). Since DEF6and SWAP-70 can heterodimerize (FIG. 6O), these results show thatinteraction of IRF5 with a SWEF heterodimer can directly regulate IRF5activity and thus indirectly alter the recruitment of T-bet to selectedtarget genes.

Example 8 Monoallelic Deletion of IRF5 Abolishes Accumulation of ABCsand Lupus Development in DKO Mice

To further evaluate the role of IRF5 in the generation of ABC cells theeffects of manipulating IRF5 levels on the in vivo expansion of ABCs inDKO mice was examined. Remarkably mice with monoallelic expression ofIRF5 (IRF5^(fl/−) DKO) exhibited an almost complete loss of ABC cells,irrespective of the markers used to identify the population (FIGS.7A-7D). Loss of ABCs was accompanied by marked decreases insplenomegaly, T_(FH) cells, GC B cells and PC cells (FIGS. 7E-7G).Further deletion of IRF5 using CD21Cre or CD11cCre to target B cells orCD11c+ cells did not result in additional decreases in the ABCcompartment (FIGS. 7A and 7B).

Monoallelic expression of IRF5 in DKO female mice also resulted in aprofound decrease in autoantibody production as evidence by bothanti-antibody (ANA) staining (FIG. 7H) and anti-dsDNA titers (FIG. 7I).Reduction in anti-dsDNA titers primarily reflected decreases in theproduction of pathogenic IgG2c anti-dsDNA Abs rather than IgG1anti-dsDNA Abs (FIG. 7I). Production of other autoantibodies likeanti-ssDNA, anti-Cardiolipin, and anti-nRNP Abs was also markedlyaffected by the loss of IRF5 (FIG. 7J). Consistent with these results,manipulating IRF5 expression also ameliorated several parameters ofrenal injury observed in DKO mice including the expansion of mesangialmatrix, the presence of hyaline deposits, the decrease in capillaryloops, and the deposition of immune complexes (FIGS. 7K and 7L). Thus,the aberrant expansion of pathogenic ABCs in DKO female mice observed invivo was dependent on IRF5 and alterations in IRF5 levels exertedprofound effects on the spontaneous development of lupus in DKO femalemice.

Example 9 DKO B Cells Upregulates Blimp1, IRF4 and CD138

While ABCs classically express IgM, the ability of ABCs to produceanti-dsDNAIgG2c upon stimulation in vitro prompted the investigation asto whether the ABCs can undergo class switching and differentiate intoplasmablasts/plasma cells (PB/PCs). Using the Blimp1-reporter DKO miceand other materials and methods described in Example 1, a population ofCD11c+ B cells that expressed high levels of Blimp1, IRF4, and CD138(FIG. 8 ) suggesting that ABCs further differentiate into CD11c+ PB/PCs.

Example 10 DKO ABCs Exhibit Sex-Specific Differences in Auto-AbProduction

One of the striking features of the lupus syndrome that develops in DKOmice is the finding that, as observed for human SLE, this disorderpreferentially affects females (Biswas et al. 2010).

Cells were sorted from aging DKO female, DKO male and Yaa-DKO male miceusing the materials and methods described in Example 1. qPCR was alsoperformed as described in Example 1.

Interestingly ABCs also accumulated in DKO male mice albeit to aslightly lesser extent than DKO female mice (FIG. 9A). Studiesdemonstrated no differences in BCR repertoire and SHM between female andmale DKO ABCs using next-gen sequencing (not shown). Unlike ABCs fromDKO male mice, however, ABCs from DKO female mice readily secretedanti-dsDNA IgG2c antibodies upon TLR7 stimulation (FIG. 9B) suggestingthat the pathogenic potential of DKO ABCs differs in female and malemice.

Yaa-DKO male mice had an increased expansion of ABCs as compared to maleDKO mice and enables them to produce anti-dsDNA IgG2a/c upon stimulation(FIGS. 9A and 9C).

Additionally, expression of cell markers on the ABCs in both DKO femaleand Yaa-DKO male mice were similar and included CXCR3, CD28, CD9, FCRL5,CD36, CD30, CD30L, c-kit, CD15, CD244, and CD68 (FIG. 9D). Expression ofIl13ra1 was also increased in ABCs from DKO female, DKO male, andYaa-DKO mice (FIG. 9E).

Taken together these results suggest that sex-specific factors regulatethe pathogenic potential of ABCs.

Example 11 Pathogenic ABCs Express Active Rho-Kinases (ROCK) 1 and 2

FoB cells from WT male and Yaa DKO male mice and ABCs from male Yaa DKOmice as well as CD11c+ plasmablasts and CD11c− plasmablasts (PBs) fromYaa DKO mice were sorted as described in Example 1 and subjected to aROCK1 or ROCK2 in vitro kinase assay (Biswas et al. 2012).

Increased ROCK2 activity was observed in ABCs and CD11c− plasmablasts(FIG. 10A). Consistent with these results, phosphorylation of IRF4, aROCK2 target, was observed in CD11c− but not in CD11c+ plasmablasts(FIG. 10B). However, in contrast to the findings for ROCK2, the samepopulations demonstrated that ROCK1 is activated at higher levels inABCs and CD11c+ PBs but not in CD11c− PBs (FIG. 10C).

Example 12 The Transcriptional and Epigenetic Profiles of CD11c+ PCs

While ABCs express both CD11c and CD11b, CD11c+ PCs downregulate CD11bexpression (FIG. 8 ) suggesting that as DKO ABCs undergo terminaldifferentiation they acquire unique characteristics. Using the materialand methods described in Example 1, a genome-wide approach is used totest the hypothesis that the transcriptional and epigenetic landscape ofCD11c+ PCs is distinct from those of ABCs and of “classical” (CD11c−)PCs.

With the aid of the Blimp-YFP reporter, ABCs(CD11c+CD11b+CD19hiCD23−Blimp1−CD138−), CD11c+PCs(CD11c+CD11b−CD19loBlimp1hiCD138hi), and “classical” PCs(CD11c−CD11b−B220loCD19loBlimp1hiCD138hi) from aging DKO female mice aresorted. As control “classical” PCs are obtained by immunizing wtBlimp1-YFP reporter mice with NP-CGG as described (Jones et al. 2016).

The different sorted populations are analyzed using will be subjected toRNA-seq and ATAC-seq as described in Example 1. These data are comparedwith previously performed genome-wide analyses (Shi et al. 2015). Aselected number of targets are validated by QPCR, Western blotting,and/or FACS.

Given that CD11c+ PCs express both IRF4 and IRF5 and that both IRF4 andIRF5 have been reported to regulate the expression of Blimp1 (Kwon etal. 2009), the hypothesis that IRF4 cooperates with IRF5 in regulatingthe differentiation/function of CD11c+ PCs is also tested using thematerials and methods of Example 1.

The IRF4^(fl/fl) DKO mice, described in Example 1 are crossed toT-bet-Zsgreen-T2ACreERT2 DKO mice. This strategy minimizes the knownimpact of IRF4 deletion on other cellular compartments like DCs (whichwould be affected by using the CD11cCre line) and “classical” B cellcompartments (which would be affected by using CD23Cre mice). While DKOmice do exhibit an accumulation of TFH cells that produce IFNγ, asmentioned above, these cells do not express T-bet and thus should not beimpacted by the removal of IRF4. All relevant control genotypes are alsoincluded.

Once these mice have been generated, IRF4 deletion is induced bytamoxifen administration (and verified by QPCR and or IC FACS) and theeffects of removing IRF4 versus IRF5 (using theT-bet-Zsgreen-T2A-CreERT2IRF5^(fl/fl) DKO mice) are evaluated byperforming a series of experiments which will include: i) a baselineFACS and serum Ig analysis of 8-12 week old mice, ii) a series ofstudies to assess PC differentiation in vitro upon exposure to differentcombinations of ABC-promoting stimuli, and iii) an evaluation of thedevelopment of lupus-like disease in aged groups of female mice asdescribed in the previous examples. If warranted, additional geneticmanipulations will also be investigated (e.g., IRF4fl/+IRF5fl/+).

A series of mixed bone marrow chimeras are also performed, which willtake advantage of the μMTDKO mice. Briefly lethally irradiated μMTDKOrecipient mice will be reconstituted with mixtures of 80% μMTDKO BM+20%of CD11c−Cre+IRF4fl/flDKO BM so that only CD11-c expressing DKO B cellswill lack IRF4. Once appropriately reconstituted, a full analysis ofyoung and aged mice is performed as described in detail above.

These experiments show a unique transcriptional and epigenetic profileof the CD11c+ PC cells from DKO mice. They also show that both IRF4 andIRF5 regulate the differentiation of these cells from ABCs as well astheir function. IRF4 and IRF5 co-regulate a common set of targets inaddition to each of them controlling separate targets.

Example 13 Sex-Specific Mechanisms Controlling ABCs

A number of observations have implicated sex-specific pathways in theregulation of ABCs. As shown in Example 10, there is a strikingsex-specific differences in the ability of ABCs to produceautoantibodies. Also, while ABCs accumulate in both DKO female and malemice, only ABCs from DKO female mice readily secreted anti-dsDNA IgG2cantibodies upon TLR7 stimulation (Example 10), suggesting that the ABCsfrom DKO females are functionally distinct from those obtained from DKOmales. Dysregulation of TLR7 expression in DKO male mice, i.e., Yaa-DKOmice, rescued their ability to produce anti-dsDNA IgG2c (Example 10).Using the materials and methods described in Example 1, using bothtargeted and genome-wide approaches, the hypothesis that sex-specificpathways regulate not only the expansion but also the function anddifferentiation of ABCs is tested.

To gain new insights into additional sex-specific pathways that mightcontrol the function/differentiation of ABCs, RNA-seq and ATAC-seq isperformed on ABC cells sorted from DKO male and Yaa-DKO male mice (andsex-matched control mice). These profiles are compared to those obtainedin ABCs from DKO female mice (Examples 4-6). A motif analysis isperformed to determine whether functionally relevant differences map tomotifs of specific transcription factors.

These studies are complemented by experiments in primary B cellspurified from young female DKO, male DKO, and Yaa-DKO male mice (andappropriately sex-matched wt controls), which are cultured invitro±ABC-promoting stimuli (which include IL-21, IFNγ, and TLR7 addedin various combinations to αIgM+αCD40) followed by evaluations of ABCformation/function/differentiation by FACS and QPCR/Western of selectedtargets as well as additional assays like ChIP-QPCR and ONPs asdescribed in Example 1.

The transcriptome of ABCs from DKO male and female mice show somesimilarities (since aberrant expansion of these cells is observed inboth female and male DKOs). The ABCS from DKO males and DKO females havecrucial transcriptional differences that is revealed by a GSEA analysisand these distinctions reflect the differential expression of a selectedgroup of pathways, and that the ATAC-seq demonstrates that thesetranscriptional differences are accompanied by differences in thechromatin landscape surrounding these genes.

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The invention claimed is:
 1. A method of treating systemic lupuserythematosus in a subject in need thereof, comprising theadministration of a therapeutically effective amount of a nucleic acidwhich encodes human DEF6 protein and a nucleic acid which encodes humanSWAP-70 protein, wherein the administration of the nucleic acid whichencodes human DEF6 protein and the nucleic acid which encodes humanSWAP-70 protein abolishes or decreases pathogenic age-associated Bcells, and wherein the pathogenic age-associated B cells are CD11c+ andsecrete anti-dsDNA IgG.
 2. The method of claim 1, wherein the nucleicacids are delivered directly to the pathogenic age-associated B cells inthe subject.